Electroejaculators,
Morphology
and
Microscopes.
General Introduction 3
1. ELECTROJECULATORS AND ELECTROEJACULATION 3
Introduction ………………………………………………………………………………………………………………………………………………………. 3
Facilities …………………………………………………………………………………………………………………………………………………………….. 3
To Bail or not to Bail ………………………………………………………………………………………………………………………………………….. 3
Simple Modifications …………………………………………………………………………………………………………………………………………. 4
Floor and foundations of crush ………………………………………………………………………………………………………………………….. 8
Talk to the owner ………………………………………………………………………………………………………………………………………………. 8
Electroejaculators ……………………………………………………………………………………………………………………………………………... 10
Maintenance of machines and probes ………………………………………………………………………………………………………………. 10
Electroejaculation Techniques …………………………………………………………………………………………………………………………... 10
Difficult bulls ……………………………………………………………………………………………………………………………………………………... 11
Yard decorum ……………………………………………………………………………………………………………………………………………………. 12
Vocalisation ………………………………………………………………………………………………………………………………………………………. 12
Sample identification ………………………………………………………………………………………………………………………………………... 12
Check list of field items ……………………………………………………………………………………………………………………………………... 14
Summary …………………………………………………………………………………………………………………………………………………………... 14
2. MORPHOLOGY 15
Spermatogenesis ……………………………………………………………………………………………………………………………………………….. 16
Stages of spermatogenesis ………………………………………………………………………………………………………………………………... 16
Factors affecting sperm production …………………………………………………………………………………………………………………... 19
The difference between compensable and uncompensable sperm abnormalities …………………………………………….. 20
The importance and timing of retesting bulls that fail their first morphology assessment ………………………………... 20
Standardisation of recommendations to the profession ……………………………………………………………………………………. 21
Prognosis from morphology reports ………………………………………………………………………………………………………………….. 21
Sperm abnormalities …………………………………………………………………………………………………………………………………………. 21
Head abnormalities …………………………………………………………………………………………………………………………………………... 21
Midpiece/tail defects ………………………………………………………………………………………………………………………………………... 22
Cytoplasmic droplets …………………………………………………………………………………………………………………………………………. 23
Testicular degeneration …………………………………………………………………………………………………………………………………….. 24
General discussion …………………………………………………………………………………………………………………………………………….. 24
Sale bulls ……………………………………………………………………………………………………………………………………………………………. 25
Commercial bulls ……………………………………………………………………………………………………………………………………………….. 25
3. MICROSCOPES 26
Mass activity and motility …………………………………………………………………………………………………………………………………. 26
Care and cleaning of your microscope ……………………………………………………………………………………………………………….. 27
Ancillary equipment ………………………………………………………………………………………………………………………………………….. 29
Microscope suppliers …………………………………………………………………………………………………………………………………………. 32
Appendix 1 ‐ Consumables ………………………………………………………………………………………………………………………………... 33
References ……………………………………………………………………………………………………………………………………………………………….. 34
Authors: Peter McAuliffe, Helen Johnston, Peter Johnston, Viv Perry © Australian Cattle Veterinarians (ACV) 2010. ACV is a special interest group of the Australian Veterinary Association Limited. Acknowledgements: The authors are indebted to Prof.A Barth for his technical consideration of and advice on this document, and to Judy Felton Taylor for her considerable assistance with the manuscript. Layout: Kay Yates and ACV Staff
2 The Australian Cattle Veterinarian • www.acv.com.au The aim of this document is not to re invent the wheel, but rather to concentrate on tools and tips for the
processes involved in assessing bull fertility. Some topics are brief where reference is given to more
detailed literature but other less documented topics are dealt with in full.
1.
ELECTROEJACULATORS AND ELECTROEJACULATION
Introduction
The most important aspects of bull fertility testing that can make the difference between a successful day
and a disaster are; (a) animal welfare (b) workplace, health and safety and (c) accuracy of data recording
and sample collection.
The Bull Breeding Soundness Evaluation (BBSE) is being rapidly accepted as a standard procedure in
beef cattle production. The most common method of obtaining a semen sample during this examination
is by the use of an electroejaculator (EJ). There are important welfare issues with this procedure, and,
although modern machines are vastly superior to those of the past, stories still abound of happenings
years ago.
Colleagues are strongly advised to read the excellent paper by Carter and McCann (2009) to be
conversant with the animal welfare aspects of electroejaculation. At the end of the day we will be judged
by how quietly we handle the bulls and how smoothly the procedure takes place.
Information from Work Place, Health and Safety places Agriculture as the most dangerous work place with cattle production, in particular, at the top of this sector. Injuries to the upper limbs account for 39% of
injuries, with 14% affecting the hands and fingers, 5% the wrists and 10% each to the elbows and the
shoulder regions. We are regularly called upon to carry out procedures in facilities that are far from ideal,
or which are, in fact, quite dangerous. We are faced with two possible actions: either we walk away from
the job or we choose to complete the task at hand. Invariably, for various reasons, we choose to carry out
the task and hope that the bulls behave, the client is satisfied, the job is completed and that we are not
injured. We should not have to work under such pressures or risks and there are often simple ways these
may be reduced.
Facilities
Facilities, or the lack of, are regarded as the most important consideration relating to EJ’s and their use.
A mini publication has been produced about cattle crushes, but the reality is that we are going to have to
use the facilities provided at the time and most cattle crushes were not designed for the electroejaculation
of bulls. (Australian Cattle Vets (2010) Crush Design & Safety)
To Bail or Not to Bail
As a general comment it is considered better not to catch the bull’s head in the bail during the
electroejaculation process. Certainly, some individual bulls will tolerate their heads being caught and will
literally stand and deliver. Should you choose to catch their heads, make sure that their heads can be
released in a hurry! This is particularly important where the bottom of the bail tapers to a V. If a bull goes
down on his knees in a bail of this configuration the situation can turn very ugly, very quickly. When the
head is not caught and the bottom gate is opened the bull will generally try to escape. With a small
number of bulls, say 1-5, you may manage by opening the bottom gate a small amount at the last minute.
Trying to operate the EJ, open the gate and collect the sample can be done but it is not without significant
risk. This technique is strongly discouraged.
Ejaculators, morphology and microscopes 3 Simple Modifications
There are a number of simple modifications that can be made to the crush that will transform a very
difficult situation into a reasonable and safer work situation. Often a bull can be collected in the sections
before or after the crush. Figures 1 and 2 show the testicles being palpated and measured in the crush
and the collection carried out after the crush.
Fig 1. The “inspection door” in the kick gate facilitates testicular measurement. Fig 2. In this situation the separation of the testicular measurements and electroejaculation makes a huge difference to the testing of these bull (n=60‐80). 4 The Australian Cattle Veterinarian • www.acv.com.au In Figure 3 the bulls were examined and collected in the section before the crush with a restraining bar
inserted at the appropriate length behind the bull. At times we may have to work without a kick gate. If
the bar is well clear of the hocks, if the bull’s movement is restricted and if he is back on the bar, we are
“reasonably” safe. This is a judgement each operator will have to make.
Fig 3. This situation before the crush was used with great success. The pipe was adjusted so as the bull was restricted. These were experimental bulls and were well behaved. There was no kick gate and the disadvantages of this needs to be considered. Generally these bull (n=22) behaved very well in this section. The actual crush can also be modified simply to facilitate the procedure. In Figure 4 the crush is longer
than average - an advantage when testing bulls. The owner made a metal frame that attaches or hangs
on the crossbeam frame that is between the two top gates and bottom gate. The frame is constructed so
as it fits inside the bottom of the crush. A rubber blanket is attached to the front of the frame.
Fig 4. Described in the text, this kick gate has a heavy duty safety chain. Kick gates can and do get kicked open. Ejaculators, morphology and microscopes 5 The bulls are walked into the crush and the kick gate is closed. The head bail is partly opened and the
bull is allowed to put his head through whilst a pipe is inserted behind him, see Figs 5 and 6.
(Very occasionally, a bull will break through the bail and will need to be brought around again.)
Fig 5. Note the high position of the bar behind the bull. The bar needs to be of sufficient length, have a well defined, limited position, and either is held or pinned. Heavy gauge chain Fig 6. This is considered to represent the ultimate in convenience and safety for testicular palpation and measurement. This is over the gate and under the bar which is further forward than the gate. Note the heavy duty safety chain in the bottom right hand corner. 6 The Australian Cattle Veterinarian • www.acv.com.au The testicles are then palpated and measured. You can see from the Figure that the bar is relatively high
up on the bull’s hind quarters and well clear of his hocks. There is ample room to work over the top of the
kick gate and under the bar. A safety chain secures the kick gate. One needs to be aware of the position
of the rail behind the bull in relation to his hocks. Unless this is properly placed, bulls, without kicking,
may still fidget and lift their hind legs, which can trap ones fore arm/wrist between their hocks and the bar.
Once the measurement of the testicles is completed, the bar is removed, the bull’s head is let out of the
bail and the bull will come back onto the kick gate. The bull is then ready for the collection, see Fig 7.
(McAuliffe, P 2002). It is an excellent example of a safe working environment.
Fig 7. The same metal insert has been used in shorter bails with similar success. Fig. 8 shows a standard
length crush where the bull is restrained with a metal bar and the kick gate is open. The distance
between the kick gate and the sliding gate behind is limited and for this reason the testicles are measured
partly from the side without getting in completely behind them.
Fig 8. The bar is not as high as in a previous photograph but this situation works well. It is important to have the bull well back on the bar. Ejaculators, morphology and microscopes 7 Fig. 9 shows the bull in the crush, his head not in the bail, the pipe still in behind him and the kick gate
open. The kick gate is then closed, the pipe removed and the bull is electroejaculated. Despite the
apparent cramped fit, quite large bulls have been successfully collected at this facility.
Fig 9. It is a matter of modifying the existing facilities to make them more workable. Professor Albert Barth (2010, pers. comm.) advocated that the front third of the crush be enclosed as
much as is possible. That is, the bail, when closed, is solid (non see through) and the front third of the top
and both sides of the crush are enclosed. This could be done with black plastic, rubber sheeting, plywood
or even cardboard. In his opinion, this almost totally eliminates fractious behaviour. For really fractious
bulls, his advice was to electro ejaculate them first and then carry out the measurements. He has found
that they will be much better behaved.
Floor and foundations of crush
The floor surface of a lot of crushes is very slippery. They come with a painted surface of high gloss paint
(presumably for rust prevention) and a pattern on the surface designed to prevent slippage. Some
surfaces have small crossbars at regular intervals to give traction to hooves. Generally speaking these
modifications are ineffective. Some crushes will also sway and give the impression of instability, the bulls
resent this movement. The positioning of the crush/foundations and the slipperiness of the floor surface
are very important and may well have a deleterious effect on our work situation and results.
Talk to the owner
As we said earlier we are going to have to deal with the facilities that are provided. In most cases the
owner is not going to go out and buy a new crush. With some simple modifications, most situations will
end up safer and more productive work places. These modifications are not expensive and it will
probably only be a matter of discussing our requirements with the owner. We all know how potentially
dangerous testing bulls can be.
8 The Australian Cattle Veterinarian • www.acv.com.au Two further examples of simple modifications are in Fig.10 - Mitchell and Fig. 11 - Millmerran.
Fig.10 Mitchell. A section of metal panel blocked the front half of the crush after the lower gate was opened. Sometimes the head was in the bail depending on the size and behaviour of the bull. A balk gate with the bail partly open will help accommodate larger bulls. Generally backward and forward motion needs to be restricted. Photo courtesy of: Anne‐Charlotte Orefice. Fig 11. Millmerran. A simple length of heavy duty ply made a huge difference to this situation. Photo courtesy of: Fergus Dance Ejaculators, morphology and microscopes 9 Electroejaculators
Over the years a number of machines have been used.
They are as follows:
1.
2.
3.
4.
Standard Precision Electronics from Denver USA.
Lane 111Z from Denver USA.
CGS from Victoria.
Lane Pulsator 1V – Auto Adjust from Denver USA.
1. Standard Precision Electronics Unit
This has been found to be a very good and compact machine. It has been used on large numbers of Bos
taurus bulls in the age range of 12 to 30 months with excellent results. Its action is smooth and quick. As
far as Bos indicus bulls and their derivatives are concerned, it has appeared to lack a degree of power,
rendering it unsuitable for the wide spread use on these breeds. It just does not seem to have enough
power for the larger bulls.
2. Lane IIIZ
This is a small light machine with an external battery. There were only about twelve of these units made.
It has plenty of power and works well. There is no facility for an external power supply. This machine has
given good service.
3. CGS
This machine has been used a lot and is a very good machine. It has an internal battery and facilities for
an external power supply. For large numbers of bulls, more than 15, it is best used with an external
power supply such as a car battery. It does have plenty of power and this should be used with caution.
There is no join where the cord attaches to the probe, this is considered an advantage. This machine will
produce an erection and will exteriorize the penis in 95% plus of Santa Gertrudis bulls. The after sales
service is excellent.
4. Lane Pulsator IV
This is a relatively new and excellent machine. It does have a long lasting internal battery and can be
used connected to mains power with an adaptor. It can be used either manually or with a preset
automatic program. Its action is smooth and effective.
Maintenance of machines and probes
As with all machines, maintenance is very important. Switches, batteries, cords and connections are the
areas where problems are likely to occur. It is important to read the manufacturer’s instructions regarding
the care of the equipment and to follow their recommendations. Batteries should be kept charged and
replaced if necessary. Breakdowns or stoppages in isolated areas can be very frustrating, especially with
a yard full of bulls. A spare ejaculator and cord are considered to be a good investment. Connections of
the cord to the machine and the probe need to be well maintained or replaced regularly. The connection
at the probe needs to be kept dry. The lead can be left attached to the probe throughout the day as long
as it stays dry. The electrodes of the probe are to be kept clean with a mild scour pad and cleaner.
Electroejaculation Techniques
The first point that needs to be raised is to read the manufacturer’s instructions regarding the use of the
machine, and to take the process gently. A second important point is that bulls must be sexually rested.
It is very difficult to draw semen from a bull that has been with cows during that past 24 to 48 hours.
Carry out a rectal examination to empty the rectum of excess faeces and to examine the accessory sex
organs in the pelvis. Massage the ampullae for a time before the probe is introduced. The introduction of
the probe can be facilitated by the retracted gloved hand maintaining an opening at the sphincter. A
person at the rear of the bull is considered essential to prevent the probe from being expelled, and if
necessary remove the probe quickly.
10 The Australian Cattle Veterinarian • www.acv.com.au The new Lane probes have the handles and power connection above the centre point which has the
effect of tilting the probe forward. One slight disadvantage of the CGS probe is that without any
“handles”, the probe may rotate slightly within the rectum with the result that one hind leg will be affected
more than the other.
Start the electro ejaculation process slowly and watch the bull rather than the controls. Start on the
lowest setting and turn the stimulation knob until the bull starts to react. Initially this may be shown by him
pulling his prepuce up a little. Hold him at the end of each stimulation for a short time and then return the
stimulation knob to zero. The time that it takes to increase the power and the holding period will be in the
vicinity of 2-3 seconds. This process will be repeated a number of times within each power level until the
maximum stimulation for the power level is attained. To change power settings, as the stimulation knob is
returned to zero, quickly move your hand across to the power level knob and adjust it up one setting and
then back to the stimulation knob and resume the stimulation. As the process progresses, the tip on the
penis will start to appear and there will be clear seminal fluid. This will progress to where all or most of
the penis is extended and the seminal fluid goes from clear, to cloudy, to semen.
It is reasonable to describe the process as trying “to draw him out”, that is - his penis and then the semen.
Play him like a fish on the end of a line. If the maximum level of stimulation has been reached (for the
CGS this is 4; with the Lane IV, normally only go to 6 or 7) and there is no semen after 3-4 stimulations,
you should stop. Be ready to catch semen as it may run out spontaneously during the rest period. At this
stage with some bulls semen can be collected by immediately turning the level of stimulation down to
either the second or third lowest setting and quickly turn the stimulation (output) knob to full power either
once or twice. If there is still no semen, stop and let him stand for a while (this may be only 30-60
seconds) and then start the process again. On the second round of stimulation you can start from the
second level of the power setting. Generally after the second round of stimulation semen will be collected.
If there is still no semen one can attempt to massage semen out of the ampullae by pressing them against
the floor of the pelvis and drawing them back towards the urethra, or depending on the size of the bull and
the probe being used it may be necessary to try a larger sized probe. If semen is still not collected, his
future is starting to become a little clouded. However, be EXTREMELY careful before you declare a bull
as being unable to produce semen.
If practitioners are experiencing difficulty with their technique for ejaculating bulls,
Prof. Barth (2010, pers. comm.) comments:
The most frequent cause of failure to protrude the penis is inadequate rhythm and rate of stimuli. Until protrusion takes place, the rate of stimuli should be rapid, lasting only about a second and setting up a rocking motion in the bull’s hindquarters. Once the penis is out, longer stimuli can be used to get semen emission. Most bulls will produce an acceptable semen sample without too much trouble; however we should not
expect 10ml of pristine semen every time we electroejaculate a bull. If a semen sample cannot be
collected after two attempts as described above, let him out and bring him back at the end of the day. If
we can do no better at the second attempt, depending on his history, I would still not condemn him on that
day. If the poor result is repeated after a period of time, his situation is looking rather grim. Only on rare
occasions will bulls not produce semen of some kind. They may repeatedly produce dilute semen; say
with a density of 1-2 (on a scale of 0-5). In making a decision on such a bull we need to consider his
morphology, his age, his scrotal circumference in relation to other bulls in the herd, their semen results
and the presence of any pathological changes in the scrotum, the region of the scrotal neck and his
pelvis. After these factors have been considered, discuss them with the owner, and decide his fate.
Difficult bulls
Most bulls will behave in a satisfactory manner. They need sufficient space but should not be allowed to
move backwards and forwards. Some bulls will be more excitable than others whilst some will try to climb
out of the crush. One of the instruction manuals described the treatment of these bulls as simply riding
them out, which is keeping up the stimulation until they settle down.
Ejaculators, morphology and microscopes 11 We agree with this to a certain degree but prefer the technique whereby, if a bull becomes unduly excited
or distressed, the stimulation should be stopped and he be let to stand for 30-60 seconds. The stimulation
is then commenced again and most bulls will eventually co-operate. Err on the side of caution and go
through the process a number of times until a satisfactory result is obtained.
Some bulls will go down as soon as any significant stimulation is applied. We need to adopt the attitude
that to have a bull go down in the crush represents a major defeat or poor handling of the situation.
Nevertheless the odd bull will go down. They generally start off by partly flexing one fore leg and leaning
against the side of the crush. At this stage, stop and get him to stand up again. The next stage is for him
to go down on one or both knees. Work through this problem in the same manner as described for
excitable bulls. Should he persist in trying to sit down or if he goes down, give the electroejaculator away
and collect a sample by massage.
Yard decorum
Try to keep the work place as quiet and peaceful as possible. Often one vet and one operator are better
than one vet and 2-3 operators. Bulls do seem to respond to being handled in a quiet and calm manner.
Do not use electric prods or working dogs. Discourage the attendance of visitors. If something is to go
wrong it will go wrong whilst they are watching and bad news spreads quickly.
Vocalisation
The experience of dealing repeatedly with large numbers of young experimental bulls would suggest that
electroejaculation is not a painful procedure. There may be degrees of moans and groans but nothing
more. Some bulls will bellow with their tongue protruding. This is considered to be due to acute and
painful seminal vesiculitis. These structures need not be greatly increased in size; however on palpation
they may have an oedematous, almost gelatinous feel when they are in the acute phase. It is considered
that their reaction does represent a significant degree of pain. Veterinarians will have to use their own
judgement when handling these cases; massage of these bulls is one option.
Sample Identification
It is very easy to make a mistake with the identification of morphology samples. Concentration is required
with the recording of identification details. A breakdown in identification can have serious repercussions.
Fig 11a . Better quality micro tubes are preferred as some have been known to come open. This one has a safe lock cap. The micro tube placed on the plastic end cone is identified on the right with 39 for the number of the bull for the day. The numbers on the left are 12.8.10 for the date. This identification makes samples “impossible” to mix up. 12 The Australian Cattle Veterinarian • www.acv.com.au A plastic or polystyrene holder for the micro tubes will greatly assist with this task.
Check your work details and after every five or ten bulls check that you have the required number of
samples. Also check that the tubes are marked correctly and that there are not two with the same
number.
Fig 11b.
Again check that you have all your samples before the bulls are let out of the yards. As added security
the disposable plastic end cones can be numbered with the collection number for that bull. See photo
above.
As a last resort these can be retrieved from the bin in cases where the bulls have been let out of the
yards.
Ejaculators, morphology and microscopes 13 Check list of field items (see Appendix 1 for details of consumables)
Microscope with phase in travel case, spare bulb
Microscope cleaning material - blower brush, lens cleaning tissue and isopropyl
alcohol, available from a compounding pharmacist
Warm stage
Warming device e.g. small incubator for keeping items warm
Thermometer digital Max/Min -20 to 50ºC, resolution 0.1ºC
Glass alcohol thermometer in metal case, -5 to 50ºC
Field book, work sheets or laptop
Electro ejaculator with cords and probe, preferably with a spare of all of these items
Disposable plastic end cones or disposable sleeves and collection tubes
PVC holder for plastic end cones/sleeves
1.5ml morphology tubes containing fixative (see appendix 1 for details)
1.5ml micro tubes with phosphate buffered saline for motility examination
Microscope slides and cover slips
Pipettes – Pasteur and or plastic transfer
Permanent marking pens plus pencil if tubes have a white writeable surface
Some form of tube holder, polystyrene or plastic for collected morphology samples
Zips lock plastic bags for secure transport of morphology samples
Power supply if required
Power board
Electric kettle
Gloves - both pregnancy testing and wrist length blue nitrile
Overalls, hat, suitable footwear
Dust masks
Summary
If our hands or other parts of our upper limbs are injured we will be unable to work. We have highlighted
ways of making our every day work situations safer and more efficient with simple, inexpensive minor
modifications to existing facilities. We need to discuss with our clients our concerns and requirements.
People are very aware of the changes in attitudes towards work place health and safety. We need to be
on the lookout for ideas to improve crush designs and communicate these to the Australian Cattle
Veterinarians (ACV) and, in turn, to the crush manufacturer. Finally we need to be aware of animal
welfare considerations and strive to carry out our work in a manner that does not cause distress to the bull
and is acceptable to our clients.
14 The Australian Cattle Veterinarian • www.acv.com.au 2.
MORPHOLOGY
Bull fertility is one of the three most important factors in determining conception at first service, the others
being a decrease in Body Condition Score (BCS) at breeding and time of previous calving (Anderson et al.
1986). The hidden economic cost of failure to conceive is often overlooked. For every 21 days of the
breeding season that a cow remains open, her calf will be 21 days younger at weaning and thereby, on
average, 25kg lighter. Basarab (1993) in a study of 2713 producers showed that when the percentage of
calves born in the first 21days of the calving cycle increased from 29% to 49% profits increased by $39/
cow.
The prevalence of infertile bulls has not changed for 7 decades. Lagerlof in 1934 found that 23% did not
pass a fertility test, Carroll et al. in 1963 20%, Barth in 1983 25% and Bertram in 1993 (pers. comm.)
20%. The percentage of all bulls that fail on semen quality range in these studies between 10-14%.
Current data (2009) from 3 sperm morphology laboratories in Australia would agree with this range.
A parameter of semen quality; sperm morphology (the structure/shape of the bull sperm) has been known
to be of importance for nearly 80 years. Williams and Savage (1927) summarized in their clinical
observations the following findings. “(1) The dimensions of sperm heads from good fertility bulls are
remarkably uniform, (2) we have observed no highly efficient bulls which emitted more than 17% abnormal
sperm, and (3) permissible numbers of abnormal sperm in an ejaculate depends largely on the type of
abnormalities present.” There have been a large number of papers written relating to the importance of
sperm morphology. Fitzpatrick et al. (2002) and Holroyd et al. (2002) published the findings of a large field
project that involved large numbers of cattle spread across northern Australia. This research was known
as The Bull Power Project. It is recommended reading for workers in this field. Phillips (2003) also
published work relating to the importance of sperm morphology and fertility. This guide therefore will
address the relationship between bull fertility and sperm morphology and the interpretation of
morphological assessment. It will also cover important aspects of correct routine bull examination and
semen collection to ensure that the test result we obtain is as accurate as possible. It is important to keep
in mind the structure of the normal sperm cell. Figure 12 shows the features of the bovine sperm.
Fig 12. Structure of a bovine sperm (Hafez 1993) Ejaculators, morphology and microscopes 15 Spermatogenesis
Spermatogenesis is a long process of cellular differentiation, in which a spermatogonial stem cell
proceeds through several mitotic divisions, a meiotic division, and numerous cytological transformations,
leading to the generation of mature elongated spermatids. Sperm cells are formed in the seminiferous
tubules of the testes. The original stem cells or spermatogonia are always situated in a layer at the base
of a tubule. They undergo several mitotic divisions to form spermatocytes, which are located a layer
higher. The spermatocytes then go through the long process of reductional or meiotic divisions to form the
next layer of haploid cells called spermatids. The spermatids, which are originally round cells, undergo a
complex series of transformations to form a final layer of elongated spermatids with a head and tail and
are then released into the lumen of the seminiferous tubules. This final cellular transformation is referred
to as spermiogenesis. Thus differentiation of the germ cells into their final form is always associated with
their upward mobility in the seminiferous epithelium. The Sertoli cell, whose cytoplasm encompasses the
germ cells, assists in the upward mobility and development of these cells by providing physical,
nutritional, and hormonal requirements. (Barth and Oko 1989)
Explanations and diagrams relating to spermatogenesis have often appeared daunting and complicated to
understand. Barth (2007) simplified the diagram by Hochereau (1967) and Berndtson and Desjardins
(1974). His diagram is presented below.
Stages of spermatogenesis
In diagram 13a the cells are always read from left to right and bottom to top as we move through the four
and a half lines of cells. The diagram has four and a half lines or cycles - actually 4.3 to 4.7 cycles
(Hochereau-de Riviers 1981) - with each cycle taking thirteen and a half days. (4.5 cycles x 13.5 days =
60 days, add 11 days for passage through the epididymis = 71 days from the start of the first cycle to the
appearance of semen in the collection tube.
Figure 13a. Stages of spermatogenesis (Barth 2007) Figure 13b. Days before each sperm will appear in the ejaculate
Figure 13b. 16.5‐11 30‐17.5 44‐31.5 58‐45.5 71.5‐59 16 The Australian Cattle Veterinarian • www.acv.com.au 1
Spermatogonia occupy the bottom one and a half lines, up to and including the seventh cell
on the second line.
2
Primary spermatocytes are represented by the last four cells on the second line and all but
the last cell on the third line. The last cell on the third line is a secondary spermatocyte.
3 The fourteen stages of spermiogenesis are illustrated by spermatids on the upper two (one
and a half) lines.
4
Each cell is 13.5 days older than the cell below it in the diagram.
5
A cross section of the wall of the tubule is represented by a vertical line though the diagram.
6 The 12 stages of each cycle take 13.5 days to complete.
The effect of an insult will depend on (a) the severity of insult and (b) the period between collection of the
sample and the time of insult.
The number of days till each abnormality appears in the ejaculate depends on the number of days of
development the sperm cell still has to undergo until it is released into the lumen of the seminiferous
tubule and the number of days the sperm cell spends travelling the length of the epididymis.
The whole process is very fascinating and we will consider the last one and a half lines of spermiogenesis
in a little more detail. During this stage the round spermatids mature and become elongated spermatids DNA becomes highly condensed, the acrosome is formed, the tail is formed and the cells become
potentially motile - these elongated spermatids move to the lumen of the seminiferous tubules. In detail;
golgi granules coalesce to form the acrosome, one centriole gives rise to the flagellum (tail), the other will
form one of the first two asters for chromosome separation in the first cleavage after fertilization. The
nucleus moves to the periphery and the chromatin condenses. The spermatid rotates so that the tail
sticks out into the lumen. The mitochondria congregate around the flagellum and become localized in the
midpiece. Sertoli cells have engulfed most of the cytoplasmic mass when the sperm are released into the
lumen of the seminiferous tubule but a small amount remains at the neck (the proximal cytoplasmic
droplet). The proximal droplet moves to the distal end of the midpiece during the epididymal passage.
This distal droplet is lost during transit in the epididymis or during ejaculation.
This process is expanded in the following table.
Ejaculators, morphology and microscopes 17 Fig. 14. Spermatogenesis and corresponding abnormalities ‐ the Roman numerals indicate the 14 stages of spermiogenesis Days of development of the sperm 61‐71 55‐60.5 XIII‐XIV 52.5‐54 XI/XII .
51.5‐53 IX‐X 47.5‐50.5 VIII‐IX 41.5‐46.5 I‐VII 28‐40.5 14.5‐27 1‐13.5 Stage of Development Abnormality Passage through the epididymis Proximal droplet moves to distal end of the midpiece. This distal midpiece distal droplet is lost here or during ejaculation. The final reflexes hormonal maturation of the sperm occurs in the distal droplets epididymis. Sertoli cells continue to engulf the cytoplasm. The fibres mitochondrial of the axoneme increase markedly in size. The midpiece‐
disturbances principal junction is formed and mitochondria condense detached above this to form the midpiece section. A small amount heads of cytoplasm (the proximal droplet) is left at the neck of the flagellum after condensation of the mitochondria. The basal plate connection between the head and midpiece capitulum is formed. The nucleus is completely flattened; the acrosomic granule knobbed is replaced by a protruding crescent shaped structure, the acrosomes apical crest. proximal droplets nuclear Nucleus flattens as the nuclear condensation increases; the postacrosomal sheath is formed over the distal part of vacuoles the nucleus. The membrane sleeve and the fibrous sheath continue to develop. coiled Nucleus elongates and starts to condense but is still round principal in cross section. The cytoplasm has moved distally so that pieces Sertoli cells now surround the head cap. A membrane sleeve and fibre bundles can be seen round the developing pyriform axoneme (the structural core of the flagellum) and heads continue down to form the principal piece. Golgi vesicle is derived from the Golgi apparatus. The acrosomic granule within this vesicle develops. A cap gradually forms over the nucleus with the acrosomic granule centred over the head of the cap. The Golgi apparatus migrates to the distal end of the head cap. The flagellum starts to grow from the distsal centriole. Meiosis continues until, by the end of the third cycle, each primary spermatocyte has produced two diploid secondary spermatocytes. These quickly divide to give four haploid spermatids to end meiosis. Spermatogonia continue dividing through two more generations to produce primary spermatocytes. The long process of meiosis begins. Golgi apparatus and more organelles appear in the cytoplasm. Three mitotic divisions of spermatogonial cells give rise to developing spermatogonia and new reserve stem cells. Days Post Insult 1‐3 4‐11 11‐15 18 20 22‐24 25.5‐30.5 31.5‐44 45‐57.5 58.5‐71 Note –The numbers in the last column are an indication of the minimum days since the insult can have
occurred before the collection of the ejaculate.
18 The Australian Cattle Veterinarian • www.acv.com.au Fig. 15. Development of the bovine sperm (Public Domain) Factors affecting Sperm Production (derived from Barth 2007)
a
Stress – pain, hunger or cold. High levels of testosterone are required for normal
spermatogenesis and epididymal function. Marked suppression of testosterone levels
can be caused by stress via increased cortisol production.
b
Heat – obesity, scrotal abnormality, climate and fever. The scrotum is normally well set
up for cooling; however, a distinct scrotal neck is required. About five metres of
testicular artery are coiled up in the scrotal neck region. Testicular function is
normally on the brink of hypoxia. With an increase in temperature there is an
increase in metabolic rate in the testis, but there is no corresponding
increase in blood flow. Tissues are therefore susceptible to hypoxia.
c
Season – photoperiod, nutrition and temperature
d
Heredity – sperm defects, testes size
e
Puberty – resembles disturbed spermatogenesis
f
Toxicity
g
Nutritional – protein, vitamin A and selenium deficiencies
Ejaculators, morphology and microscopes 19 The difference between compensable and uncompensable sperm abnormalities
Much research remains to be done in determining the levels of the various sperm defects that can be
tolerated in the naturally mated bull. The defects are categorised according to the possible effect the
abnormality may have on fertility, based on the current literature. The terms primary or secondary are no
longer used as these were based on where the abnormality occurs rather than the effect upon fertility.
The ACV has adopted the terminology put forward by Evenson et al. (1999) who considered that semen
may contain sperm with both compensable and uncompensable traits. Understanding the compensable/
uncompensable concept is simple if we consider that the female tract (Mitchell et al. 1985) and finally, the
vestments of the ovum act as a filtration system for the sperm population. They act as barriers to the
progress of sperm such that only the fittest arrive at the ovum (Saacke et al. 1988).
Compensable traits preclude affected sperm from fertilizing the ovum, i.e. the abnormality does not allow
them either to reach the ova or attach to the ova. A compensable abnormality, therefore, is one that can
be compensated for by increasing the number of sperm in the ejaculate; that is, the fertility of the bull will
increase with increasing numbers of sperm. They include traits that cause abnormal or nil motility (sperm
are filtered out in the female tract) and abnormal head shape (sperm are filtered out crossing the zona as
this interferes with hyperactive motility which is required at this juncture). Such sperm have little effect if
inseminated with sufficient normal sperm in an ejaculate.
Increasing numbers of sperm, however, cannot compensate for uncompensable traits. Sperm with these
traits are able to reach the ovum and initiate fertilization (thereby blocking polyspermy). If any embryo
development is possible, that development is unsustainable and the cow therefore returns to estrus.
Such traits include nuclear vacuoles and pyriform heads. These abnormalities tend to be more subtle and
more difficult to detect, yet they cause the most decrease in conception rates. By conducting sperm
morphology differentials in this manner we can achieve a standard and accurate prognosis of the bull’s
current performance.
The importance and timing of retesting bulls that fail their first morphology
assessment
Given the length of spermatogenesis (61 days) and of epididymal passage (10-12 days), the sample we
examine is a biopsy of testicular function that was initiated 10 weeks previously. We can at times identify
when an insult has occurred from what abnormalities are present. The ability to identify and differentiate
sperm abnormalities is, therefore, essential. The timing of retesting depends upon the abnormalities
present. This is because we can infer from the abnormalities present when the insult occurred; therefore
we can infer (knowing the length of spermatogenesis and transit) when the bull may recover and thus
when he should be retested.
When we retest, we are usually trying to assess whether this is a defect normally present in this bull or
whether it is a transient defect caused by an environmental insult. This is also true of abnormalities
indicating that the bull is immature, although, we would also, in such a case, need to consider (a) the BCS
of the bull, (b) the age of the bull and (c) the breed of the bull, taking into the account the different age of
puberty of different breeds.
It is common practice for sale bulls to be fed a grain ration prior to sale. This has been shown in some
cases to reduce the % normal sperm. There is evidence to show that the incidence of this is due to the
grain feeding itself - when feeding is sufficient to increase fat in the neck of the scrotum and thereby
reduce the ability of the testes to thermoregulate. Recent research, however, has shown that a change in
environment (penning as opposed to range feeding for example) may be sufficient to significantly reduce
% normal sperm (Callaghan et al., unpublished data).
This leads us to the problem of when to recommend to producers the optimal time for the BBSE prior to
sale. Some producers now wish the BBSE to be conducted prior to the bulls being put onto feed.
Interestingly, a recent series of experiments has established that some bulls can be assessed relatively
early, post puberty and that their semen profile remains fairly stable (Callaghan and Perry, 2010).
Whenever they are tested, the results must be a true reflection of what the bulls will be like at the time of
sale. The reputation of both the veterinarian and the bull vendor must be considered.
20 The Australian Cattle Veterinarian • www.acv.com.au Standardisation of recommendations to the profession
All sperm morphology examinations must be done under oil immersion at x 1000-1200 (Barth and Oko,
1989). Semen is collected into vials at the crush side. The amount of semen put into the vials is
dependent upon the concentration of the semen collected - the more dilute the sample, the more semen
must be added to the vial of buffered formal saline. Phase contrast microscopes are cheaper than DIC
and just as precise, if one is able to take time over the sample, except for semen extended in milk.
Therefore, both are used by accredited morphologists.
The use of stains in the examination of sperm is not recommended in the ACV accreditation system as
this method increases the number of abnormal heads and detached heads compared to wet preps
(Sprecher and Coe 1996). There is also a significant decrease in the number of intact acrosomes due to
differences between the pH and osmotic balance in the stain compared to seminal plasma
(Harasymowycz et al. 1976, Chenoweth et al. 1984). Subtle abnormalities are more easily detected in the
formal saline (FS) fixed samples than in the stained samples. If, for example, one has a bull with small
vacuoles, the morphologist using FS will find them but the one using nigrosin eosin (NE) may not - leading
to different counts. Therefore the standard recommended by the ACV accreditation scheme is FS wet
prep under oil immersion.
Prognoses from morphology reports
Evaluation of the spermiogram should be undertaken by those who understand the processes of normal
and abnormal spermatogenesis as well as the normal and abnormal ultrastructure of the sperm (Barth
and Oko, 1989). It follows from this statement that we should be able to give some prognosis from the
laboratory findings. This task is made easier for all concerned if the full BBSE sheet details are available
to both the laboratory and the practitioner, particularly when difficult cases arise.
Sperm abnormalities
A full interpretation of each abnormality is available in the 2002 Darwin proceedings - we therefore
present here only a “field guide” to each commonly found abnormality.
Head Abnormalities
Pyriform heads
Narrow in the postacrosomal region. Young bulls up to 2 years old and in good condition display a
greater likelihood of recovery from this condition than older bulls. This condition is particularly seen in
young over fat bulls. Uncompensable, >20% is not acceptable.
Knobbed acrosomes (KA)
This is an abnormal pathogenesis that may be heritable or may be due to disturbances in testis
thermoregulation (e.g. over fat, illness, toxicity, nutritional deficiency). This abnormality can be tolerated
at levels up to 30% as the knobbed acrosome sperm do not attach to ova and no zona reaction will take
place. The ova will still be available for fertilization by other normal sperm in the vicinity, thus it is a
compensable defect. This is supported by work by Andersson et al. (1988) who found that when this
abnormality was present in less than 25% of sperm there was no decrease in fertility. Interestingly, there
is a lesser form of KA - the flattened acrosome - which is recorded separately as this appears to yield
normal conception rates in a single sire mating yet is not competitive in a multiple sire mating situation.
Vacuoles and Diadem Defects
This defect occurs during spermiogenesis and can be caused by such insults as extreme temperatures or
stress. The bull can recover within 6 weeks of the insult, although some bulls are more susceptible to this
condition (probably due to a hormone imbalance in the testis) and may not recover. Based on published
Ejaculators, morphology and microscopes 21 work (Barth and Oko 1989; Olley 2001; Thundathil et al. 1998; Pilip et al. 1996), a bull with an ejaculate
containing more than 15 - 20% of sperm with significant craters or diadem defects is of questionable
breeding value as most of the literature considers this an uncompensable. Small apical vacuoles (SAVs)
are differentially listed in the comments section of the BBSE as they are more transient than diadems or
confluent vacuoles.
Swollen Acrosomes (SA)
This problem can be associated with “rusty load/accumulated sperm” and the precautions should be taken
when collecting the semen to check that there is adequate motility in the ejaculate. Ageing of the sperm
causes the acrosome to undergo a reaction similar to capacitation, resulting in the lifting of the acrosome,
and clearly the sperm will not attach to the oocyte. Swollen acrosomes are often seen in conjunction with
other head abnormalities such as knobbed acrosomes. This is because this abnormality causes
premature initiation of the acrosome reaction (Thundathil et al., 2001). In these cases spermiogenesis
has obviously been disrupted. If an ejaculate contains 70% sperm with swollen acrosomes then the bull
is considered a questionable breeder, although at only 30% he should probably still be fertile. This
abnormality is therefore compensable.
Detached Heads
This is a problem that can arise with testicular degeneration, hypoplasia, inflamed ampullae or epididymis,
heat stress and, more usually, as a sign of a “rusty load”. In Guernsey bulls, detached heads can be an
inherited problem. If the motility is low in the initial crush side motility assessment of the semen, then
further ejaculates (up to 3) should be taken so that sperm that may have “accumulated” in storage can be
eliminated and a representative sample collected. In the representative sample, the bull can still be
considered “fertile” with 30-40% of this defect but if the ejaculate contains 70% of this abnormality the bull
would have severely decreased fertility. This is considered to be a compensable effect; these sperm
cannot participate in fertilization as they cannot swim up the female tract.
Midpiece/Tail Defects
Mid piece and tail defects are generally considered to be compensable traits, as the sperm with these
traits cannot reach the fertilization site. The midpiece is the engine room of the sperm, creating motility.
Distal Reflex Midpieces (DRMs)
Not to be confused with simple bent tails, this is by far the most common defect seen in bull ejaculates. If
this defect is due, for example, to epididymal cold-shock or transient heat stress, recovery is likely within
16 days. The presence of a cytoplasmic droplet at the tail bend identifies the problem as one of distal
reflex. This abnormality occurs mainly in the distal half of the cauda epididymis. The prognosis varies
with circumstance and the types of other abnormalities present. Where it occurs with abnormalities such
as a fracture at the tail bend, aplasia of the midpiece or Dag-like defects, the prognosis is worse as there
is probably an underlying cause such as disturbed spermiogenesis.
Some bulls have a predisposition for this defect with fluctuations in the percentage of affected
spermatozoa throughout the year. Up to 30% of this abnormality is tolerated in the ejaculate as these
cells display reverse motility and would therefore be unable to penetrate the zona pellucida (Barth and
Oko 1989). This is a compensable defect.
Dag-like defect or doubly bent tails
This can be an inherited defect with a poor prognosis if present in large numbers (i.e. >50%). It can
reflect disturbance in the testicle or epididymis and is not normally present at >4%. It is a compensable
trait as the sperm are not forwardly motile. Fertility is therefore only impaired once >25% of this defect is
identified in the ejaculate with less than 70% normal sperm (Barth and Oko 1989).
22 The Australian Cattle Veterinarian • www.acv.com.au Segmental aplasia of the mitochondrial sheath
Usually only present in a small % of sperm. Johnson (1997) quotes a study by Barth (1992) in which a
bull with 90% affected sperm had normal fertility. This would indicate that the condition has little effect on
fertility. This condition can be permanent or transient; if the defect is seen to occur in two tests done ten
weeks apart it suggests a permanent condition. The condition would be compensable as it does not
affect the nuclear material; therefore some latitude is given when analysing the sample. However, it
should not be present at levels higher than 30% with 70% normal sperm according to Barth and Oko
(1989) as the affected midpiece is often fragile and susceptible to breakage.
Principal Piece Defects
These are seldom seen in high numbers and may be due to temperature shock to the epididymis,
therefore levels of this defect may decrease after 8-11 days. Levels of 30% are acceptable with 70%
normal sperm. This is a compensable abnormality.
Abaxial Tails
The level of infertility caused by this abnormality varies. The prognosis is determined by the other
abnormalities seen with it in the ejaculate. Ejaculates containing 60-100% sperm with abaxial tails cause
no decrease in fertility (Johnson 1997). However, sperm with abaxial tails seen in an ejaculate with other
sperm with accessory tails can cause a significant drop in fertility. This being the case, abaxial tails
should not be considered a defect if present on their own but if abaxial tails are present at relatively low
numbers (12-20%) with >17% accessory tails, the bull would be considered of questionable fertility (Barth
and Oko 1989). A compensable defect - in some cases not considered a defect but a normal form for
particular bulls.
Tail Stump Defect
This condition is hereditary and has a poor prognosis. However, it is a compensable defect as the sperm
cannot journey to the fertilization site and bulls with 30-40% of this defect have been found to be fertile. It
should be noted that care should be taken to differentiate this from detached heads (Johnson 1997).
Cytoplasmic Droplets
It is most important to differentiate between proximal and distal droplets when assessing this abnormality
as they have quite a different relationship with fertility in the bull. The issue has been recently raised that
distal droplets that are not associated with the DRM condition should be considered due to the
relationship between cytoplasmic droplets and ubiquitin levels. Discussions with the researcher who has
published most in this area (Peter Sutovsky) reveal that he agrees that there is not a case so far to justify
the inclusion of distal droplets (without DRMs) in the BBSE. Current research into this area is ongoing
(Perry et al. 2009 unpublished data).
Proximal Droplets (PD)
These are normal in the pubertal bull with incidence decreasing with age (Amann et al. 2000). In the
mature bull they indicate abnormal spermiogenesis (and/or epididymal function) and are often seen in
conjunction with other abnormalities of the head and mitochondrial sheath. Bulls with this defect can
recover within 6 weeks but the prognosis depends upon the type of abnormalities associated with the
proximal droplets. Even counts of 10-15% proximal droplets have been associated with decreased
motility and decreased fertility (Johnson, 1997). Sperm with proximal droplets are less able to bind to
ova. When percentages of PDs are high (>20%), even the normal sperm associated with the PD
sperm have a reduced ability to bind to ova. This trait is considered uncompensable as some sperm
are able to fertilize to the ova but cause early embryonic death.
Ejaculators, morphology and microscopes 23 Testicular Degeneration
This is a common problem that can be either (a) temporary, followed by regeneration or (b) permanent,
followed by fibrosis. The upper layers of cells in the seminiferous tubule can slough off and end up in the
lumen. Only the more robust sperm and the Sertoli cells that are closer to the blood supply on the
basement membrane will survive, the testes will become soft and no semen will be produced. When the
insult is removed, the testes may repair with regeneration and repopulation of the tubular epithelium; in
this case the tone of the testes will return to a normal or a near normal state. When the insult is more
severe and/or prolonged there can be a more serious destruction of the tubular epithelium and fibrous
tissue may be produced. This process is not fully understood; actual semen production will be very
significantly reduced but the bull can have good morphology. (Barth, Coffs Harbour 2007)
General Discussion
After having read and considered the research data, the question that we still have to answer is – what do
we tell our clients? Probably one of the most critical research findings is from the Bull Power Project
(Fitzpatrick el al. 2002). It found that in general, bulls with < 50% normal sperm sired few calves, while
bulls with the highest calf output had > 70% normal sperm, and that the percentage of morphologically
normal sperm in ejaculates was moderately to highly repeatable. Figure 16 below from the same paper
shows the relationship between sperm morphology and fertility score. It puts the question of <50% normal
morphology, 50 – 70% and > 70% of normal sperm into some perspective.
This figure also demonstrates the multi factorial nature of reproduction. It also emphasises that
morphology examination is a critical part of every BBSE whether the bull is being examined pre-sale or
pre-mating.
16
(Fitzpatrick el al. 2002 )
24 The Australian Cattle Veterinarian • www.acv.com.au As a general comment we need to bear in mind that it is not our decision as to which bulls are kept or
rejected, but rather to guide our clients through the decision making process. We need to consider the
different situations for both sale and herd bulls.
Sale Bulls
With these bulls we have the ACV guidelines for sperm morphology in terms of total normal sperm and
the percentages of maximum abnormality for each category to guide our discussions. Bull breeders are
becoming more aware of the adverse financial and good will effects of having sale bulls returned on
account of poor semen quality.
Some preliminary data (Callaghan and Perry, 2010), shows that when monitoring semen quality from prepuberty to 22 months of age, there was a small percentage (10%) of bulls whose sperm morphology was
always unsatisfactory. At the other end there was a group that always produced semen with excellent
sperm morphology, while in the middle was a group whose morphology results fluctuated significantly
depending on the stressors applied to them.
Taking young potential sale bulls from their natural grazing situation and confining them to pens, yards of
various sizes or small paddocks for the purposes of pre sale feeding and preparation can have a very
significant effect on their sperm morphology. In the experimental situation, the effects of the injection of
dexamethasone to bulls (or the insulation of their scrotums) are well documented. In experiments by
Barth & Bowman (1994) normal bulls and a precise stress or agent was used for a defined period. In the
field (practice) situation the stressors may be mixed and of varying degrees of severity or
subtlety; they may be present for varying periods of time and it might not be obvious that they are
being applied.
In separate work Callaghan et al. (unpublished data) relocated 22 adult bulls (>24 months) 200kms and
housed them in individual out door pens that were partly covered. The transport, penning and inclement
weather which followed caused a significant decline in the average percentage of morphologically normal
sperm. The challenge for us is to identify the 10% of bulls with major problems, the group that will
withstand most insults and the group that will fluctuate as pressure is applied. Unfortunately we cannot
do this with one test.
Commercial Bulls
In the case of commercial bulls the situation is a little different. Again it is not our role to direct producers
as to which bulls to use, but rather to discuss the results with them and guide them through the decision
making process. Whether the bull is in a single or a multiple sire situation and the likelihood of a
dominance effect will also need to be considered – for example, a less fertile dominant older bull should
not be multiple sire mated with a fertile younger bull. The number of bulls available for selection as well
as available finances to purchase replacement bulls often influences the decision.
The selection of herd sires will be determined by the percentage of normal sperm, the totals of the
categories of abnormalities, age, body condition, general health and physical problems, the soundness of
the reproductive organs in general and the presence of any specific pathology along the reproductive
tract, in particular in the tail and head of the epididymis, seminal vesicles and ampullae, and the freedom
of reproductive disease. In the single sire situation high standards will need to be adhered to. A multiple
sire situation should not be used as an excuse to include sires with poor credentials in the hope that they
may pick up a few cows here and there as the dominance effect, previously mentioned, may cause a
significant decrease in conception rates.
Selected bulls will need to be ready for immediate use. History relating to annual sire testing results and
owner knowledge regarding individual sires will assist selection. There is a need to minimize the
variables and strive for high reproductive performance with an emphasis on economic returns to meet the
challenges of modern day agriculture.
Ejaculators, morphology and microscopes 25 3. MICROSCOPES
We take note of David Galloway’s words of wisdom that “there was the bull, the operator and the
equipment - we need to make sure that the latter two are up to speed and if there is still a problem it will
probably BE THE BULL.”
We have, therefore, highlighted aspects of equipment or operator error that may influence the crushside
bull fertility test.
It is essential that all our equipment is adequate and well serviced before we undertake the complicated
and serious business of examining bulls for breeding soundness.
Veterinary practitioners are under constant pressure to purchase additional items of equipment or to
upgrade the equipment that they already own. Cattle practitioners are also under pressure to contain
their fees as best they can. As a consequence, cattle vets have tended to let their field microscope drop
down the priority list. This has become very obvious when cattle vets gather for semen work shops or
field days.
A good quality microscope is considered to be a good investment. Microscopes are generally classed as
student, laboratory or research. We consider that it is fair to say that there is no such thing as a good
cheap microscope. The ability of a microscope depends on the quality of the objective. A good quality
student microscope is very suitable for our work.
If you are serious about testing bulls, be prepared to spend a reasonable sum on the field microscope and
LOOK AFTER IT. Reputable brands include Zeiss, Olympus, Leica and Nikon. It is possible to get a
good estimate of motility using a microscope with only bright field, however, phase contrast will give you a
much clearer view of motility, and there is really no comparison. Crush side motility can be examined by
using phase contrast with either 20x or 40x objectives. Mass activity or swirl rate can be examined with
an objective between 3.2x and 10x magnification. New microscopes will range in price from $3,000$7,000. At the time of purchase you will need to obtain a precise price.
A second hand quality microscope can often be a cheaper way of entering the field and a representative
of the various companies may be able to direct you as to where to purchase a suitable microscope.
Microscopes can also be upgraded with phase contrast or different objectives after purchasing second
hand BUT it is always wise to ask a servicing engineer if this is possible with the model you are
considering, prior to purchase. A 40x objective and phase ring may only cost $700-$1000. This is
perhaps the most cost effective way of getting a good field microscope. We have had experience of this
with older Olympus and Leitz microscopes.
It is essential to also purchase a strong travel case. (Hardigg Storm Cases make excellent cases once
they are fitted out with foam; Pelican cases are waterproof and easy to move around on wheels.) Even if
well packed in its case, do not leave the microscope to fall from side to side in the back of the utility secure it if you want it to keep working.
Mass activity and motility
The first segment of the semen microscopic examination is to examine the mass activity or wave motion.
This can be done with a 10x eyepiece and an objective that may range in power from 3.2x to 10x. The
microscope light will need to be adjusted by closing the field diaphragm. Mass activity is a function of the
concentration and the motility. Place a small drop of semen on the warm slide and examine the sample
without a cover slip. If the sample is very concentrated draw the drop out a little with the end of the
pipette as it is placed on the slide. Look at the edge of the drop for the best estimate of the mass activity.
It is important to differentiate between concentration and mass activity, what we are looking for is the rate
at which the swirls/lines of sperm move, independently of the concentration. A glass Pasteur pipette will
deliver a smaller more controllable drop than a plastic disposable pipette.
26 The Australian Cattle Veterinarian • www.acv.com.au Motility assessment is an integral part of the BBSE examination; it is normally carried out by using a 10x
eyepiece and either a 20x or 40x objective. It is, vital that the examination is conducted professionally and
correctly, using good equipment. It is considered ESSENTIAL that a warm stage be used to accurately
assess sperm motility. All items and solutions used for this examination must be kept warm at around 37
º C with a suitable warming device. Motility can only be assessed by viewing the forward motion of
individual sperm cells. It is the percentage of rapidly moving, forwardly progressive individual sperm cells
per field, (moving at > two body lengths per second) - not those that are swimming backwards, in a circle
or moving slowly. Put simply, how many cells in every 10 are exhibiting this activity.
When semen is dilute the motility can be examined by placing a small drop on a slide under a cover slip.
In cases where the semen is more concentrated it must be diluted first before the motility can be
assessed. Semen is normally diluted by using phosphate buffered saline although sodium citrate or
normal saline can also be used. The motility of the diluted semen should be proportional to the mass
activity. Every step that we take there is always the possibility that something will go wrong, if this
appears to be the case repeat the process. If using two micro tubes for the examination, one for the
phosphate buffered saline and one for the formal saline for morphology, be very careful that cross
contamination does not take place. The opinion is that a very small volume of formol saline will go a long
way in terms of suppressing motility. Some bulls, when collected with an EJ, will have semen with motility
where the sperm cells appear to be moving uniformly at three quarter pace in a normal forward motion.
This is noted with either raw or diluted semen and is not considered to be a problem, provided a warm
stage is being used. All details regarding the motility examination should be recorded.
Care and cleaning of your microscope
Following the purchase of a new microscope it is essential that the manufacturer’s instruction
regarding care and cleaning be followed closely.
The following section has been adapted from a booklet “The Clean Microscope” published by Zeiss.
Clean microscope optics are a prerequisite for successful microscopy and perfect images. The closer
that the dirt is to the object being examined, the greater will be the effect on the image. Some optical
surfaces are more sensitive to dirt than others; the front lens of the objective is particularly critical. If the
image sharpness or contrast is not optimal, then there is a high probability that the microscope optics are
not clean.
Cleaning agents and procedures
The goal is to completely remove dust and dirt without leaving any residue of the cleaning agent or
damaging the surface. The following equipment is required.

Long, thin wooden sticks, preferably of bamboo, available from Chinese restaurant suppliers, or the
uncovered end of a long handled swab (15 cm) match stick thickness.

High purity cotton (e.g. that used in ophthalmology, supplied by KERMA Germany) or WHATMAN
lens cleaning tissue 105. The latter is available from Labtek or other scientific supplier.

Absorbent polyester swabs for cleaning optical components. ITW Texwipe Clean Tips swabs
(Texwipe), represents a very good alternative to the cotton swab.

Soft cosmetic cellulose tissue (e.g. Kim Wipes soft, KLEENEX.)

Dust Blower

Freshly prepared solution of a couple of drops of a washing up liquid in 10 mls of distilled water

Solvent for the removal of greasy or oily dirt, such as the Optical Cleaning Solution L (85% pure
petroleum ether, 15% isopropanol).
Ejaculators, morphology and microscopes 27 Preparation of the cotton swabs
Wash hands first. Dip the end of the stick or the long handled swab in the cleaning solution first; this will
make it easier to attach the cotton wool or the Whatman lens cleaning tissues. Have the cotton wool in a
zip lock plastic bag, open the bag, insert the stick and rotate it sufficiently to attach sufficient wool to make
a swab. Alternatively the lens cleaning tissue can be used in a similar fashion. This can be cut into either
halves or quarters and wrapped around the end of the stick.
Cleaning procedure (except for the optics)

Blow all loose dust particles away with a dust blower, this is VERY important.

Remove all water-soluble dirt with distilled water; if this is unsuccessful repeat using a solution of
diluted washing up liquid. Remove any remaining residue with a dry cotton swab but breathe on the
surface first to generate a layer of moisture.

To remove oily dirt use a solution of dilute washing up liquid initially. If this is not successful repeat
the cleaning using a solvent. Consult the manufacturer regarding the use of any solvent.
Cleaning procedure – optics
The use of water on lenses is not recommended as it can promote fungal growth that can be very hard to
remove.

Use a small amount of isopropyl alcohol on a lens cleaning tissue - it will give a bright smudge free
finish.

Cleaning is achieved by using a spiral motion from the centre to the rim. Never clean a lens with a
zig zag motion.

After cleaning check the surface. This can be done with a magnifying glass 3-6x or by using an
eyepiece held the wrong way round.
Avoid Contamination
Set up in a dust free area and keep covered when not in use. Covering in the field may affect the
operation of the warm stage. In tropical areas steps will need to be taken to protect the lens from fungal
contamination, this is a very serious problem.
The external surface of the microscope can be cleaned by using a soft haired brush kept exclusively for
this purpose and a lightly moistened microfiber cloth.
Practitioners may be reluctant to take a new microscope into the area of a cattle yard, select an area that
will be as dust free as possible, however, there will always be plenty of dust around. One of the authors
has a microscope that has spent 27 years in and out of dusty cattle yards, has been regularly serviced
and has suffered no long term effects from the dust.
28 The Australian Cattle Veterinarian • www.acv.com.au What to Watch Out For When Cleaning Microscope Optics!
1.
When starting to clean, don’t forget to use a dust blower, except when fluids such as immersion oil
are to be removed.
2.
Never wipe lens with dry swabs or tissues – these causes scratches.
3.
Do not use abrasive material e.g. leather wipes, dry linen cloths or polystyrene sticks as
recommended by some manufacturers.
4.
Do not use any solvents before trying distilled water (a film of distilled water can be generated by
breathing on the surface), except where grease is to be removed.
5.
Do not use ethanol or acetone for the cleaning of older microscopes.
6.
Do not use any disposable swabs apart from those already listed.
7.
Do not use any of the optical spray cans containing pressurized liquid air. This product can leave a
slight, but difficult to remove residue.
8.
Never use acids or ammonia to clean objective front lenses.
9.
Never try to clean internal optical surfaces.
Ancillary Equipment
Warm Stage
A warm stage is considered essential for correct motility assessment. Warm stages vary in price from
$400 to $2000 depending on their attributes. It is important to make sure that the “hole in the centre” is as
small as possible yet allows light through from the condenser. A large “hole “means that the slide is not in
contact with the warm surface of the stage whilst you are viewing the semen.
Warming Device
Some device, such as a small incubator, hot plate, esky and hot water bottle, thermos or water bath, is
necessary to keep solutions, items of equipment and semen warm.
Power Supply
Microscopes and warm stages can be run from the car battery. It is sometimes more convenient however
to have a small generator. Honda makes excellent quiet and reliable generators with 1 KVa output that is
quite sufficient. The same machine can also be used to power ultrasound etc. Field setup includes a
small reliable inverter generator capable of running microscope, warm stage and laptop, or an inverter
(500watts) will run all your equipment off the battery including ultrasound ($150). Select a wind free/
protected, glare free site for microscope setup
Ejaculators, morphology and microscopes 29 Field Laboratory
The facilities available “crush side” can have an influence on the quality and enjoyment of our work. On a
lot of properties they are far from satisfactory. Some simple improvements can make a significant
difference.
This is a very good crush side work area. It is protected by a wall in the north eastern corner of the
facility. Adequate bench space and a water sink help make work more enjoyable. This is part of a larger
complex where the lead up race, crush and scales are under cover. The semen examination area is
directly behind the last panel of race before the crush.
Photos courtesy of: Sonya Greenup This small shed and water tank stand was manufactured by the Davey Group an Engineering business in
Toowoomba for their own property. It is not expensive but very effective. Water from the sink discharges
to the ground. Simple improvements such as this will have very significant effect on our ability to perform
our work.
30 The Australian Cattle Veterinarian • www.acv.com.au The front compartment of a covered goose neck trailer. Known as “The Laboratory” it is taken from one
set of yards to another. Power is supplied by a portable generator. A horse float and table would also be
suitable substitute.
A table and seat inside an open goose neck trailer under a large shed offers comfort and protection from
the sun and wind.
Ejaculators, morphology and microscopes 31 A good setup in the back of a vehicle ‐ Photo courtesy of Peter Johnson Microscope Suppliers
For ease and completeness we have requested the various microscope companies to provide us with
information about their various items of equipment and their prices. This has been supplied in electronic
format that has been posted to the ACV web site.
The prices supplied were current at the time of posting - up to date prices will need to be obtained at the
time of purchase.
Useful Contacts: Mark Stafford, Zeiss 0432487076 ‐ for sales and servicing Peter Hanson, Leica 0418514988 for servicing Allan Smith, Minitube 03 53420833 for sales Windsor Davies 07 33411217 for servicing Mike Burnstein, Scientific and Optical 07 33560233, sales and servicing Coherent Scientific 08 81505200 32 The Australian Cattle Veterinarian • www.acv.com.au Appendix 1
Consumables
Disposable plastic end cones for semen collection are available from:
Minitube Victoria, 03 53428688
Standard laboratory items are available from a number of laboratory equipment suppliers, one such
supplier is:
Labtek Pty Ltd, Brisbane, phone 1300881318
Pasteur pipettes 145 mm non sterile and unplugged - pack of 250.
Transfer pipettes 1 ml or 5 ml draw, non-sterile, pack of 500.
Plain microscope slides and cover slips
Tubes safelock (Eppendorf) 1.5ml plain, pack of 1000.
Tubes micro tube (Axygen) 1.5ml, pack of 1000.
Tubes cryovials (Greiner) 2ml pack of 1000
Tissues - Lens cleaning 100 x 150 mm pack of 25 Wallet Whatman
Buffered Formal Saline*
Buffered formalin 10% clear - available from hospital suppliers.*
Dust masks - 3M 9322 from BOC
* There is some confusion with the name 10% formalin (or formalin 10%). It is a 10% solution of
saturated formalin which contains 37-40% formaldehyde depending on whether the saturation point is
measured by weight or by volume. Buffered formalin 10% clear contains 3.6% formaldehyde, a similar
concentration to that of the buffered formal saline.
(Any suppliers or contacts mentioned in this publication do not have any affiliation or commercial
relationship with Australian Cattle Veterinarians. This information is for member information only and to
the best of our knowledge accurate at the time of printing and could change.)
Ejaculators, morphology and microscopes 33 References
Australian Cattle Vets (2010) Crush Design & Safety , Queensland, Australia.
Amann R.P., Seidel G.E. and Mortimer R.G. (2000) Fertilizing potential in vitro of semen from young bulls
containing a high or low percentage of sperm with proximal droplets. Therio. 54:1499-1515.
Anderson, R.S., Fillmore, H.L. and Wiltbank, J.N. (1986) Improving reproductive efficiency in range cattle:
An application of the O'Connor Management System. Theriogenology.Aug;26(2):251-60. PubMed PMID:
16726190.
Andersson, M. Vierula, M. and Alanko, M. (1988) Acrosomal morphology and fertility in AI bulls. In Proc.
11th Int. Conf. Anim. Reprod. and AI. Paper no. 222 Dublin.
Barth, A.D. and Bowman, P.A. (1994) The sequential appearance of sperm abnormalities after scrotal
insulation or dexamethasone treatment in bulls. Can. Vet. J. 34:93-102.
Barth, A.D. and Waldner, C.L. (2002) Factors affecting breeding soundness classification of beef bulls in
Saskatchewan. Canadian Veterinary Journal 43;274-284.
Barth, A.D. and Oko, R.J. (1989) Abnormal morphology of bovine spermatozoa. Iowa State University
Press, Ames.
Barth, A.D. and Bowman, P.A. (1994) The sequential appearance of sperm abnormalities after scrotal
insulation or dexamethasone treatment in bulls. Can. Vet. J. 35:93-102.
Barth, A.D. (2007) . ACV/ARV Coffs Harbour Conference electronic proceedings.
Basarab, J.A., Rutter, L.M., Day, P.A. (1993) The efficacy of predicting dystocia in yearling beef heifers II
Using discriminating analysis. J. Anim. Sci. 71:1372 – 1380.
Callaghan and Perry, (2010) Restricted pre-weaning nutrition suppresses reproductive development in
beef bulls. Proceedings Association of Applied Animal Andrology, Sydney
Carroll, E.J., Ball, L., Scott, J.A.,(1963) Breeding soundness in bulls: a summary of 10,940 examinations.
J. Am. Med. Assoc. 42:1105–1111.
Carter M. and McCann G. (2009) Animal welfare aspects of electroejaculation. Proceedings of The 2009
Australian Cattle, Sheep and Reproduction Veterinarians conf. (Gold Coast). p69
Chenoweth, P.J., Perry, V.E.A. and Davies, C. Acrosome assessment in fresh bovine semen. (1984) 10th
Int. congr. Anim. Reprod. and AI. Illinois, USA p52-54.
Evenson, D.P. (1999) Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects.
Reproduction, Fertility and Development.11:1-17.
Fitzpatrick, L.A., Fordyce., G. McGowan, M.R., Bertram, J.D., Doogan, V.J. DeFaveri, J., Miller, R.G., and
Holroyd, R.G. (2002) Bull Selection and use in Northern Australia. Part 2. Semen Traits. Anim. Reprod.
Sci., 71 pp39-49.
Fordyce, G (ed.) (2002) Bull Fertility: Selection and Management in Australia. Australian Association of
Cattle Veterinarians Darwin Conference.
Hafiz, E.S.E. (1993) Reproduction in Farm Animals (6th Ed) Lea and Febiger, Philadelphia.
Harasymowycz, J., Ball, L. and Seidel, G.E. (1976) Evaluation of bovine spermatozoal morphologic
34 The Australian Cattle Veterinarian • www.acv.com.au features after staining or fixation. Am. J. Vet. Res. 37:1053-1057.
Hochereau-de Riviers, M.T. (1981) “Control of spermatogonial reproduction.” in Reproductive Processes
and Contraception Ed. KW McKern p 307-356.
Holroyd, R.G., Doogan, V.J., De Faveri, J., Fordyce, G., McGowan, M., Bertram, J.D., Vankan, D.M.,
Fitzpatrick,L.A., Jayawardhana, G.A., Miller, R.G., (2002) Bull selection and use in northern Australia. Part
4. Calf output and predictors of fertility of bulls in multiple-sire herds. Anim. Reprod. Sci., 71:67–79.
Johnson,W., (1997) Significance to bull fertility of morphologically abnormal sperm. The Vet Clinics of
North America, Food Animal Practice.
Lagerlof, N. (1934) Morphologische untersuchungen über veranderungen im spermbild und in den hoden
bei bullen mit vermindeter oder aufgehobener fertilitat. Acta Pathol. Microbiol. Scand. Suppl. 19: 254.
McAuliffe, P. (2002) Darwin Conference (poster presentation).
Mitchell, J.R., Senger, P.L., Ronsenberger ,J.L. (1985) Distribution and retention of spermatozoa with
acrosomal and nuclear abnormalities in the cow genital tract. J. Anim. Sci. 61:956-967.
Olley, T. (2001) Nuclear vacuole defects in bovine spermatozoa. Honours thesis, University of Queensland.
Phillips, N.J. (2003) Examination of the relationship between in vitro measures of the function of frozenthawed spermatozoa and field fertility achieved using semen collected from sires within the Australian
dairy and beef cattle industries. Ph.D Thesis, University of Queensland.
Pilip, R., Campo, M.R., Barth A.D., Mapletoft R.J (1996) In vitro fertilizing characteristics of bovine
spermatozoa with multiple nuclear vacuoles; a case study. Theriogenology 46 no.1:1-12.
Saacke, R., Nebel, R., Karabinus, D. et al. (1988) Sperm transport and accessory sperm evaluation.
Proceedings of the 12th Technical Conference on Artificial Insemination and Reproduction, Columbia.
Saacke, R.G., DeJarnette, J.M., Bame, J.H., Karabinus, D.S. and Whitman, S.S. (1998) Can spermatozoa
with abnormal heads gain access to the ovum of artificially inseminated super- and single-ovulating
cattle? Theriogenology 50:117-128.
Sprecher, D.J. and Coe, P.H., (1996) Differences in bull spermiograms using eosin-nigrosin stain, fuelgen
stain and phase contrast microscopy methods. Theriogenology 45: 757-764.
Sutovsky, P., Moreno, R., Ramalho-Santos, J., Dominko, T., Thompson, W. and Schatten, G. (2001) A
putative ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in
the mammalian epididymis. J. Cell Sci. 2001; 114:1665–16.
Thundathil, J., Palasz A. T., Barth A.D. and Mapletoft, R.J., (1998) Fertilization characteristics and in vitro
embryo production with bovine sperm containing multiple nuclear vacuoles. Theriogenology 50:328-333.
Thundathil, J., Palomino, J., Barth, A., Mapletoft, R., Barros, C., (2001) Fertilizing characteristics of bovine
sperm with flattened or indented acrosomes. Anim. Reprod. Sci. 67:231-243.
William , W.W. and Savage, A. (1927) Methods of determining the reproductive health and fertility of bulls:
a review with additional notes. Cornell Vet. 17:374.
Ejaculators, morphology and microscopes 35 36 The Australian Cattle Veterinarian • www.acv.com.au