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Artificial Insemination Vol. 1 • No.

2 • 2003 Technical Update

Artificial Insemination:
Semen Processing and Quality Control
The objective of a boar stud is the efficient production
of adequate quantities of quality semen doses of
appropriate genetics, shelf- life and health. Achievement
of this objective demands practical expertise and a high
degree of attention paid to the details. Every process on the
boar stud must be described to ensure standardization of
excellent practice. A number of studs have no w achieved
ISO9002 certification. This involves a great deal of work, but
is a clear demonstration of commitment to quality assurance,
and provides a framework for ensuring maintenance of the
highest standards.

Semen processing laboratory


The size and layout of the laboratory will vary according to the size
of the stud, collection schedules, processing routine and level of automation. The main
functions of the laboratory are:
• Storage and preparation of all equipment and consumable items required for
semen collection and processing.
• Preparation of semen extenders.
• Semen evaluation and extension.
• Semen cooling, storage, packaging and dispatch.
• Water purification
• Sterilization of equipment.

Ideally, the AI lab should be close to the semen collection area, and fitted with pass-
through windows so that semen may be transferred rapidly. On larger studs, where this is
not possible, semen may be delivered to the lab via a pneumatic tube delivery system.

The laboratory should be designed with labor efficiency and work flow in mind. While
providing adequate work surface and storage, the lab needs to be compact, to minimize
the distance between work stations, and also the amount of cleaning.

A well-designed AI lab should have:


• Washable, non-slip floors, walls and work surfaces.
• Plenty of storage space.
• Heating, and air-conditioning (where appropriate).
• Adequate lighting, avoiding any source of artificial UV light.
• Adequate electrical sockets for every item of lab equipment (surge protectors for
some items of equipment may be necessary).
• Hot and cold running water.

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• A system for the preparation of purified water for semen extension (unless this is
to be purchased).
• A means of sterilizing re- usable items (e.g. an autoclave).
• Laundry for clothes (where the AI Center is sufficiently large).
• Fly/insect remover and/or killer.
• Provision of a back-up generator
• Backup drinking water supply for the boars.

Consumable items
Semen collection, evaluation, processing and dispatch utilize large quantities of single-
use disposable items e.g. collection bags and/or cups, filters, gloves, plastic pipettes,
microscope slides, tubing, extender bags and/or vats, dispatch boxes and/or bags. These
must all be of high quality and adequate for the purpose, and are best purchased from a
reputable AI equipment manufacturer. Whilst it may be tempting to reduce costs by
purchasing a lower grade cheaper alternative, great care must be taken to ensure that
anything used will not damage the semen. You must check that each item is non-
spermicidal. You must also consider the quality of an item and weigh this against the
cost. Finally, you need a reliable source of consumables, and have a back-up plan in case
there is ever a problem with supply.

Lab computer software


In the early days of AI, labs had to keep paper records of every stage of the process, from
the semen collection rota through to the number of doses sold per boar per ejaculate.
This situation has been enhanced greatly by the development of computer software
designed especially for the running of the stud and AI lab. The level of complexity of the
software package used depends upon the size and scale of the AI operation. It is worth
discussing your needs with the AI equipment manufacturers (see list below) at an early
stage when planning a stud.

Semen evaluation
Every aspect of AI demands a high degree of quality control and consistency. A fixed
protocol must be followed in order to ensure semen of consistently high quality. Semen
should be examined immediately after collection. Throughout semen processing, every
effort should be made to avoid rapid changes in temperature. Remember that all
processing steps are a balance between speed and accuracy.

Volume of the ejaculate


Weigh the ejaculate on an electronic balance in order to estimate the volume (1ml semen
weighs approximately 1g). This indirect method of measuring semen volume is gentle to
sperm cells, and is sufficiently accurate for the purpose.

Sperm count
This is usually measured using some form of photometer calibrated for the purpose,
which is quick and easy to use. Two popular models are the MicroReaderT M (IMV) and
the SpermacueT M (Minitube). The major disadvantage of any photometric method of
estimating sperm count is that it measures optical density only, and cannot therefore

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differentiate between live and dead sperm, agglutinated sperm etc. A more accurate
method involves counting the actual number of sperm cells using a cell-counting chamber
slide (e.g. Neubauer or Burker chamber). Although far more time-consuming, and rarely
used for routine work, the counting chamber is useful for calibrating the photometer, and
carrying out periodic checks on the number of sperm per dose.

Motility estimation
This provides a measure of sperm viability. Motility may be evaluated for the raw
ejaculate, for partially and fully extended semen. It provides an estimation of the
proportion of sperm cells showing progressive forward motility. This parameter is
important because it has an impact on the subsequent fertility of the dose.

Prepare a microscope slide by warming it to approximately 950 F (350 C). Place one
standard-sized drop of semen onto the slide using a pipette, and place a warm cover slip
over the drop. Use a microscope fitted with a warm-stage at 950 F (350 C) and examine
the sperm cells at x200 or x400 magnification to estimate the level and vitality of sperm
movement.

Examining raw semen, it is possible only to give the quality of sperm motility a score
according to a simple scale (e.g. from 1-5, where 5 is excellent, 3 is acceptable for further
processing, less than 3 to be discarded).

Where partially or fully extended semen is examined, attempt to estimate the proportion
of sperm that are active, and the quality of that activity, rating it in terms of the
percentage of sperm showing progressive forward motility. Note that in extended semen
samples, it is easier to visualize individual sperm cells than in a raw ejaculate as the
density is lower. In this case, a minimum of 60% motile sperm is essential for good
levels of fertility with AI.

Some of the larger studs in the US are now adopting Computer Aided Semen Assessment
(CASA) systems such as IVOST M from Hamilton Thorn Research, or SpermVisionT M
from Minitube. These systems utilize computer technology to track individual sperm
cells, evaluating various parameters of their movement (e.g. direction, velocity, angle of
curvature between the head and the tail) and can provide a much more accurate, objective
and repeatable measure of both sperm count and motility. They represent a significant
investment initially, and also require a commitment to train staff properly, but yield
benefits in terms of accuracy in semen evaluation, reduction of sperm wastage and labor
saving in the long-term.

Sperm morphology examination


Assessing the shape of sperm cells in an ejaculate is time-consuming, and rarely
conducted on every ejaculate from every boar. It should be used according to some kind
of routine however e.g. once a month for boars with good semen quality, more often for
problem boars, and as a useful test for new boars. It involves examination of a stained
smear of sperm cells at x1000 magnification (using an oil emersion objective). Eosin-
Nigrosin or Trypan Blue are examples of the stains that can be used for this purpose.

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Mix a few drops of raw semen with the required amount of stain in a test tube (see
individual manufacturers’ instructions for exact quantities), incubate at 98-1000 F (37-
380 C) for 5-10 minutes, prepare a smear on a microscope slide and allow to dry.
Alternatively, mix the semen and the stain together on the slide, and prepare the smear
directly. Examine a minimum of 100 sperm cells across the slide at x1000 oil emersion.
Assess the morphology of each sperm, recording which ones are normal and which are
not (see diagram for sperm morphological types). For AI use, and ejaculate must have at
least 75% morphologically normal sperm cells.

Some of the computerized systems for semen evaluation can also classify sperm
morphology.

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Other Tests
There are a number of other more complex tests available for assessment of semen
quality that focus on sperm function e.g. the osmotic resistance test (ORT), acrosome
integrity evaluation, vital staining. At this stage, they are not in routine use on most
studs.

A word about new AI boars


Pay special attention to the semen quality of new boars. Detailed semen assessment
should be carried out on the first three ejaculates collected, including raw and diluted
motilities, sperm count, volume, viability on storage, and sperm morphology. Ideally, the
initial tests should take place whilst the boar is in isolation. Non-return rates, which
should be monitored continually, are the first indication of the actual fertility of an
individual boar. Progeny inspection provides vital information with reference to
congenital defects.

Semen extension
Semen should be extended as soon as possible after collection. Most studs aim at
a sperm dose of 2.0-4.0 x 109 viable sperm in 75-100mls. extender. The number of doses
made from an ejaculate is calculated as follows:

No. normal sperm/ml. x Volume of ejaculate(mls)


No. sperm required per dose

For example:
No. sperm per ml semen: = 0.525 x 109
Volume of ejaculate = 120 mls
No. sperm required per dose: = 3.0 x 109

No. doses: = (0.525 x 109 ) x 120


3.0 x 109

= 21

The quantity of extender to be added to the ejaculate is calculated as follows:

(No. sperm doses x Volume of each dose) – Total volume of the ejaculate

For example:.
No. semen doses: = 21
Volume of each dose: = 90 mls
Volume of ejaculate = 120 mls

Volume of extender = 21 x 90 - 120

= 1770mls

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Having calculated the dilution rate, and the required volume of extender, add the extender
to the ejaculate, making sure that their temperatures are the same. The target extension
rate (by volume) is 1:10 to 1:20. Extender can be added all at once, or in two stages.
After extension, assess sperm motility, expressing it as a percentage of motile sperm – it
must be at least 60% motile on the day it is used. Decrease the temperature of the semen
from 98-1000 F (37-380C) to a storage temperature of 59-640 F (15-180 C) gradually over 1-
2 hours.

Preparation of pooled (heterospermic) semen


Pooled semen is a mixture of semen from two or more boars. Some large-scale studs
pool semen in order to simplify dispensing large numbers of semen doses, particularly
when an automated dispensing system is in use. There is conflicting evidence as to
whether fertility with pooled semen is better than that of the individual ejaculates used
separately. Whenever pooled semen is used, it is important to make sure that the semen
from each contributing boar is of good quality. Pooling semen will not compensate for
poor semen quality – in fact, quite the reverse is true. A poor ejaculate mixed with a
good one can bring down the overall quality of the pool. When pooling semen, the
following steps are essential:
• Check the motility of each ejaculate.
• Fully extend each ejaculate in the same type of extender, and keep a sample of
each in the lab for quality control purposes.
• Carefully mix together the extended ejaculates, making sure that they are at the
same temperature when you do this.
• Check the motility of the pooled semen after mixing. Keep a sample of the pool
in the lab for quality control purposes.

Semen Extenders
The functions of the semen extender are to expand the volume of the ejaculate, and to
maintain sperm cell viability. Extenders may be prepared from raw ingredients in the AI
laboratory, but this is not recommended. High precision weigh-scales are required in
order to do the job properly; doing the job yourself may compromise quality control, and
is not cost effective. Most AI studs purchase their semen extenders in powder form.
There is a wide range of semen extenders available commercially, which vary in
performance and price. They include BTS, MerckIIIT M, VitalT M, AndrohepT M, XcellT M,
MR-AT M, Androhep EnduraguardTM and SafeCellT M.

The choice of extender depends on the shelf- life required, and the price you are prepared
to pay. Whilst the formulation of many extenders is now either patented or confidential,
it is important that you have some information about this from both health and safety, and
quality assurance viewpoints. Also, it is essential that you are clear about the antibiotic
content of the extender you are using. If any problem should arise regarding the
bacteriology of the semen doses you may need to alter the antibiotic content of the
extender, and this is impossible without knowing what is added to start with.

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Whatever extender is used, it is essential to record the batch number, and to observe the
manufacturer's recommendations with regards to storage conditions, shelf- life and
preparation. Ensure that your supplier adheres to a rigid quality control protocol, as any
mistakes will have a direct impact on performance.

Bacteriologal checks of extended semen


Regular bacteriological screening of extended semen samples should be part of the
quality control protocol of any stud. Semen extenders generally contain high levels of
glucose, and this provides an ideal environment not only for sperm cells, but also any
bacteria that might have contaminated the semen. Bacteria produce toxins as they
multiply and die, whic h will affect sperm survival. Bacterial contamination of the semen
can occur at any stage, and great care should be taken to minimize the risk by
establishing the critical control points in the process (Quality Assurance schemes are
designed to fulfill this function). The antibiotics included in the extender formulation
should deal with any minor contamination that does occur, but are no reason for allowing
hygiene standards to slip. Plan to send samples of extended semen to a specialist
bacteriology lab monthly. Aim to maintain a clean record of semen bacterial count, but
do not overreact if the occasional bacterial colony is identified in a single dose. If this
should happen, send further samples before reviewing procedures. If a problem cannot
be solved by upgrading hygiene standards throughout the process, it may be necessary to
change the antibiotic content of the extender in consultation with your veterinary advisor.

Testing semen for presence of pathogens


The main benefit of AI is the exploitation of high genetic potential by using superior
boars more widely than would be possible through natural service. This can result in the
semen from a single boar being used on more than one farm on a given day, and presents
challenges in terms of stud biosecurity. If a boar has a viral infection when semen is
collected, there is a chance of virus being present in the semen doses. Whilst every care
needs to be taken to avoid introduction of infections into the boar stud, it is possible for
boars to become infected and to start shedding virus into the semen before the onset of
other clinical signs of disease. In the case of PRRS, an infected boar may shed virus for
several weeks.

Development of new diagnostic tests for the presence of virus in semen have been
developed. PCR (Polymerase Chain Reaction) technology is extremely sensitive, and can
identify presence of very low levels of virus in a sample. The PCR test for PRRS virus in
semen works well, and many studs now send semen samples for testing on a routine
(weekly) basis. The turnaround time for the test is rapid, with results being available
within hours of receipt at the lab. Where PRRS is an issue, it would be possible to hold
semen doses in storage for 24 hours, dispatching to farms only after a negative PRRS
PCR test result has been confirmed.

Water quality
A dose of semen is at least 90% water! The quality of water used for the preparation of
the semen extender is very important, but the choice of processes can be confusing. In
simple terms, the following water purification methods are appropriate:

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• double-glass distilled
• a combination of reverse-osmosis, de-ionization, carbon filtration, and exposure
to UV light

Water may be purified in the AI lab, and this is common practice for larger studs. The
best method for your laboratory will depend upon the quality of the raw water at the point
of delivery. AI equipment manufacturers can provide guidance on the type of processing
plant best suited to your individual requirements. Where purified water is purchased,
make sure you are satisfied with the process employed, and the quality control adopted
by the supplier. Note that the quality of purified water deteriorates with time, and
according to the method of storage. When purchasing purified water, make sure that no
more than 1 month’s supply is on site at any one time, to avoid deterioration of water
quality.

Preparation of semen the extender


Extender powder should be stored in accordance with the manufacturer’s instructions
(usually “in a cool, dry, dark place” or “at 40 C”). Observe the “use-by” date on each
packet/container, and rotate supplies to ensure that the oldest extender is used first. It is
worth recording the batch number of each container of extender, in case any problems
arise. The semen extender should be mixed with the required volume of purified water
on each day semen is being collected and processed, allowing 60 minutes for
equilibration before use. Make sure that both the required weight of extender powder,
and the required volume (or weight) of water are measured accurately, so that the liquid
extender is of exactly the right concentration (osmolarity). It can help to warm the water
to 100-1040 F (38-400 C) prior to mixing, as this speeds up the dissolving process. Once
prepared, the semen extender should be used within 24 hours.

Extender vats are available for larger-scale operations, with a capacity of 100+litres.
They are made of stainless steel, usually lined with a disposable plastic liner, and are
fitted with a mixer and a thermostatically-controlled heater. They can be installed onto a
weigh-cell to simplify filling with the required amount of purified water. Usually the vat
is filled the day before, and the heater set to switch on during the night, ha ving water at
the correct temperature in time for semen processing the following morning. Extender
powder is then added at the start of collections, allowing time to equilibrate before use.

Semen dispensing and dispatch


Extended semen is dispensed into insemination bags or tubes either by hand (on smaller
studs), or more often using an automated packaging system. Systems are available that
fill, seal, label, sort and record doses from individual boars and semen pools. This
technology has revolutionized semen processing, and made significant labor savings (for
further details contact the equipment manufacturers listed below).

Once semen has been dispensed into doses, it must be cooled to 59-640 F (15-180 C) prior
to dispatch. The cooling process should be gradual (1-2 hours from dispensing into bags
or tubes), to avoid damaging the sperm cells.

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Semen storage
Semen should be stored at 59-640 F (15-180C) until use, and protected from U-V light.
Gentle rotation of the semen doses twice daily while in storage allows the sperm cells to
be re-suspended in extender - this appears to enhance semen preservation. All stored
semen should be checked daily for sperm motility before use by incubating a small
standard volume of diluted semen (e.g.1ml) at 980 F (370 C) for a standard period of time
(e.g. 10-20 minutes).

Equipment manufacturers

The equipment and consumable items mentioned in this text is available from:
IMV
11725 95th Avenue North
Maple Grove, MN 55369, USA
Phone: 763-488-1881, Fax: 763-488-1888

IMV
BP 81 61302 L’Aigle Cedex, France
Phone: +33 2 33 34 64 64, Fax: +33 2 33 34 11 98

Hamilton Thorne Biosciences, Inc.


100 Cummings Center, Suite 465E
Beverly, MA 01915, USA
Phone: 978-921-2050, 800-323-0503, Fax: 978-921-0250

Kubus SA
Calle E, s/n
Pol. Ind. C/E Europolis
28230 Las Rozas
Madrid, Spain
Phone: +34 1 636 0268, Fax: +34 1 637 5313

Minitube of America
P.O. Box 930187
Verona, WI 53593, USA
Phone: 800-646-4882, 608-845-1502, Fax: 608-845-1522

Minitub GmbH
Hauptstrasse 41
84184 Tiefenbach b. Landshut, Germany
Phone: +49 8709 9229 0, Fax: +49 8709 9229 39

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PIC USA
PO BOX 348
Franklin, KY 42135
www.pic.com/usa
1-800-325-3398

Copyright 2003, PIC USA, Inc. All rights reserved.


A Company

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