BRAHMAN NEWS DECEMBER 2015 ISSUE #189 PAGE 24

By Ced Wise on behalf of Rocky Repro

This article is based on a presentation Ced made at a forum sponsored by Rocky Repro at CQLX, following the ABBA Annual General Meeting.

Above all else, a bull is bred and sold to produce calves.

The most common definition of a fertile bull, as distinct from one that is capable of producing a pregnancy, is that a fertile bull should be capable of getting 60% of normal cycling cows in calf in the first three weeks of mating and 90% within nine weeks (pregnant at 42 days). To achieve this, the bull must be, broadly speaking, able to seek out the oestrus cow, capable of having an erection and causing intromission and finally ejaculating viable semen into the anterior vagina.

The sperm cells present in an ejaculate are produced in the testicles and this paper briefly discusses:

• The physiology of sperm production;
• The measurement of testicular function;
• The limitations of those measurements; and
• Some of the factors known to adversely affect sperm production.

It does not examine the many other factors affecting fertility such as; physical structure (e.g. feet, legs, prepuce and penis), libido or dominance.

SPERM PRODUCTION

Testicular tissue is primarily composed of millions of very small tubes called seminiferous tubules.

These tubules form loops that flow into the centre of the testis, emptying into the rete testis.

In cross section (Figure 1), these tubules are lined on the base with round cells each containing a full set of chromosomes (30 pairs). Between the base cells and the sperm cells, on the luminal edge, scientists have concluded there are 12 columns of cells each with big names going through (to me) an incomprehensible process. The complete process takes 60 days, in this time the cell obviously changes shape dramatically (from a round cell to a long cell with a motile tail), but also halves the number of chromosomes to 30 single chromosomes.

This drawing is super simplistic, just how simplistic can only be imagined when it is known that the average bull produces around six thousand millions sperm cells every day. There is a lot happening in this highly active tissue and unless the workplace is air-conditioned, production will decline or perhaps even stop.

Testicular air-conditioning is called thermoregulation.

THERMOREGULATION

The temperature of the blood coming out of the body of a healthy bull heading towards the testicle in the testicular artery is 38.5°C. By coiling the vein that exits the testicle around the artery, the cooler venous blood cools the arterial blood temperature by 4°C. This complex arrangement between the artery and the vein, which is commonly called “the cord”, is known as the pampiniform plexus (Figure 2), and is responsible for maintaining the testicular temperature at 4°C below normal body temperature.

The venous blood is cooled because the testicles are hung in the breeze (simple physics) and evaporative cooling lowers the temperature of the blood in the veins of the scrotal skin.

The subcutaneous dartos muscle and cremaster muscle that run between the testicle and the body wall combine to raise or lower the testicles depending on the ambient temperature (nearer the body and warmer, away from the body and cooler) (Figure 3).

SPERM TRANSPORT (LOADING THE GUN)

Once the sperm cells are produced in the testicles they are transported via the epididymis (head, body and tail) and vas deferens to the ampulla. The ampulla is effectively the storage area in the pelvis,

FIGURE 1: SEMINIFEROUS TUBULES CROSS-SECTION

Source : Pathways to Pregnancy and Parturition, Second Edition – P.L. Senger

FIGURE 2: PAMPINIFORM PLEXUS

Source : Pathways to Pregnancy and Parturition, Second Edition – P.L. Senger

FIGURE 3: CREMASTER MUSCLE & DARTOS MUSCLE

Image provided by Rocky Repro

accumulating sperm prior to ejaculation. As already noted sperm production in the testicles takes 60 days. See Figure 4.

The trip through the epididymis of some 120 feet takes 10 days and during this time sperm cell maturation is completed. Sperm cells leaving the testis are not capable of fertilisation.

It is important to note that the epididymis must have high levels of testosterone to function correctly.

It takes another two to three days for the sperm cells to get from the epididymis to the ampulla (via the vas deferens), where they are stored ready for ejaculation.

The testicular function in the majority of bulls, at least in younger bulls is good but some bulls for whatever reason are infertile. These bulls need to be identified and culled. Identifying these infertile animals requires that the functionality of testicles be measured.

MEASURING TESTICULAR FUNCTION

Currently there are three tests regularly used to measure testicular function:

1. Scrotal circumference;
2. Sperm motility (mass activity and percent progressively motile); and
3. Sperm morphology.

1. SCROTAL CIRCUMFERENCE

Scrotal circumference is measured (Figure 5) because it is:

1. Highly correlated to testis weight, which is highly correlated to daily sperm production (about 10 million per gram, per day) and sperm quality;
2. Moderately to highly heritable;
3. A more accurate predictor of age of puberty than age or weight;
4. Highly correlated to the age of puberty of his heifer progeny; and
5. Easy to measure and repeatable.

2. SPERM MOTILITY (CRUSH SIDE)

Sperm cells have to be motile. They have to do a lot of miles – cervix to egg is about 600mm, which does not seem a long way but for a sperm cell that is only 60um (1/15mm) long (from the top of his head to the tip of the tail) that is a long way. Admittedly, there is a little help from uterine contraction but the sperm cell needs to be motile.

There are two ways of assessing motility:

• Mass activity; and
• Progressive motility.

To assess the mass activity, a drop of semen is placed on a microscope slide and examined at 100 magnification.

The gross/ mass activity or swirl is subjectively graded on a scale of 1-5, where 1 has no swirl and 5 is extremely active.

The mass activity of a sample is determined by:

1. The concentration of the sperm cells;
2. The percentage of motile sperm cells; and
3. The speed at which they are swimming.

The score given for mass activity has to be interpreted with these variables in mind. A semen sample with a mass activity of 4 for example, will have to have a good concentration with a high percentage of motile sperm cells that are fast swimming. Whereas a dilute sample may also have a high percentage of motile sperm cells swimming fast, but because of a lower

FIGURE 4: sperm transport

Source : Pathways to Pregnancy and Parturition, Second Edition – P.L. Senger

FIGURE 5: measuring scrotal circumference

concentration (possibly due to operator error or prior ejaculation) the mass activity will be low.

The second way of assessing motility is to look at the percentage of progressive motile cells in the sample. Here a cover slip is placed over a drop of semen, creating a single layer of cells. These are then examined at 200 or 400 magnification and the percentage moving forward is subjectively assessed.

It should be emphasised that both of these assessments (mass activity and percentage motile) are subjective, and therefore subject to the inherent individual variations that are seen in all such tests. What one person sees as 80% motile, another might see as 70%. The Australian Cattle Veterinarians now accredit vets that have demonstrated they have the equipment and knowledge to perform these examinations. This is an attempt to reduce this variability, but human nature dictates that some variation will always be present.

3. MORPHOLOGY

Morphology looks at the anatomy of individual sperm cells. This is done by placing a couple of drops of semen into formalin to kill and preserve the sperm cells, allowing them to be examined at 1,000 magnification. This cannot be done “crush side” as it requires a very large and specialised microscope that cannot be safely transported. 100 cells are counted, noting normals and abnormals, and describing the abnormals. The results are reported as the percent of normal and the percent of each type of abnormality found.

These abnormalities can be broadly grouped into compensable and uncompensable defects (Figure 6).

In simple terms, sperm with compensable defects will never reach the egg and therefore will not interfere with fertilisation by a normal sperm. That is, these defects can be somewhat compensated for provided enough normal sperm are present. Uncompensable defects on the other hand are capable of getting to the egg and blocking access to it by normal sperm, however, are then not capable of producing a pregnancy.

Please note motility does NOT equal morphology.

There is a difference between motility and morphology. A normal sperm maybe dead and an abnormal sperm may well be swimming. This explains how it is possible to have 40% progressive motility and 90% normal (Figure 7) or 90% progressive motility and 40% normal (Figure 8).

SEMEN TESTING (PREDICTING FUTURE FERTILITY)

In my opinion, the tests just described allow a reasonably good assessment of the fertilising ability of the ejaculate examined, but the goal of a “semen test” is, in reality, an attempt to predict the fertility of the bull for the next 12 months or more.

In the following comments, the parameters by which the bulls future fertility is assessed are listed along with some of the background research that was used to set these parameters.

It should be stressed, that the interpretation of this data and how it applies to an individual bull is my personal view only.

SCROTAL CIRCUMFERENCE

The acceptable size varies with age; however, all bulls in good order should be no less than 34cm at two years of age. Maybe a little less than this for a Bos Indicus bull because of their different shape.

The testicular size of the adult is determined by genetics and pre-weaning nutrition, but it should be remembered, that there is around 2-3 cm difference in the scrotal circumference of a bull measured when fat versus in working order.

MOTILITY

Progressive motility has to be at least 60% and most preferably 70% or better to allow semen to be successfully frozen.

Probably because of this, the current recommendations are that a bull should have above 60% progressive motility to be considered for use in a single sire situation. Or above 30% to be considered for multiple sire duties. BUT, having said that, the only evidence that I know of, suggests that 30% is satisfactory and that grading bulls whose motility is above this is not reflected in his fertility. This may just be a reflection of the natural variability seen between collections and the difficulty of preforming this “crush-side” examination in any repeatable way but that is the data.

Regardless of where the bar is set, (and for my two cents worth, I agree with the current settings) in my opinion there is way too much variability in the measurement to accurately discount a bull that has 10% or even 20% lower motility than another. That is, a bull with 75% motility is not necessarily worse than one with 85% motility, they are both acceptable.

MORPHOLOGY

This subject has now become the most hotly debated topic around the cattle yards, replacing banning live trade and climate change.

FIGURE 6: abnormal sperm cells

FIGURE 7: dead and normal

FIGURE 8: motile and abnormal

Proponents of morphology testing believe it is critical to the assessment of a bulls fertility and that it is positively correlated to the fertility of the herd in general. Opponents believe it to be of no value at all.

In my opinion, many of these claims are either exaggerated, not yet proven or flat out wrong, certainly under pre-sale testing conditions.

Let me state from the beginning that I absolutely believe the morphology versus fertility story OF an ejaculate. This is why Rocky Repro does a full morphology assessment on every ejaculate before freezing semen.

There has been much work done looking at the effects on fertility of different levels of all sorts of defects. These researchers have basically concluded that an ejaculate should ideally have greater than 70% normal sperm cells and certainly greater than 50% to have good fertility.

This depends a lot on the type of defect – compensable / uncompensable.

There is also some Australian evidence suggesting that similar parameters might be applicable in the field at least for Bos Indicus derived bulls (Bull Power, Fitzpatrick et al., 2002). Each bull was given a fertility score based on the number of calves he sired as compared to the average of the group.

The data showed that in the group of bulls that had 70% or greater normal morphology, 70% were above average fertility and 30% below average.

In the 50-70% normal morphology group, 25% of the bulls were above average fertility and 75% were below average.

And in those bulls that had less than 50% normal morphology there were 0% with above average fertility.

The conclusion being that if all the herd sires on the one property were tested and those with below 70% were culled there would be an increase in the herd fertility.

If you look at the individual bulls that make up this data set rather than the herd trends, I would contend that 25% of bulls in the 50-70% normal group were better than 30% of the 70+% normal group. Furthermore, those in the below 50% normal group were no worse than the 30% below average bulls in the 70% normal group.

I interpret this data more along the lines of “the probability” that a bull with between 50-70% normal having an above average fertility is not as good as one having 70% normal or better. That is a lot different to saying he will not have good fertility.

When predicting the future fertility of a bull, as distinct from the fertility of a single ejaculate, the line at 70% is really just a convenient line in the sand and should be open to interpretation based on such things, as the individuals performance within his contemporary group.

There are numerous examples of bulls that have had one lower than optimal morphology test result but have had normal fertility in the following year.

One reason for this is in the variability seen in the bull’s morphology samples overtime and even between ejaculations on the same day. The data in Figure 9 shows morphology results of a bull at Rocky Repro, from March to July 2015. The results reported on the same day, are sequential collections taken on that day.

For example, the first collection on the 2/6/15 had a Qualified Pass (QPass) at 68%, the second collection that day was a Pass at 76%, and the third collection a QPass at 68%.

In my opinion, morphology testing of bulls will eventually have to either rank them within contemporary groups, where a series of tests are conducted and the conditions are replicated (e.g. nutrition, handling, collector, morphologist). Or probably more accurately, rank sires based on the morphology result of their sons.

When this is done it may well be found that all the positive traits currently attributed to morphology testing by some are valid, but when doing a single pre-sale test I think the best that can be said is that at this time, for this ejaculate this bull was able or unable to produce a high percentage of normal sperm cells.

FACTORS DETRIMENTAL TO SPERM PRODUCTION

No doubt there are both genetic and environmental components involved in sperm production. It is certainly known that scrotal circumference is heritable and we may well find in time that morphology is too but I will leave that story to the geneticists and discuss briefly some of the environmental factors that can affect sperm production.

The major factors affecting sperm production are:

1. Inadequate thermoregulation;
2. Stress; and
3. Physical damage.

1. INADEQUATE THERMOREGULATION

1. Scrotal fat;
2. Fever;
3. Environmental heat; and
4. Anatomical.
5. What better way to raise the temperature of something than to wrap it in a layer of fat. Works well for polar bears and seals.

FIGURE 9: batch morphology

1. It is well known that having bulls fat does not do their semen any good and it is equally well known that if they are not fat they won’t sell. Furthermore in our part of the world getting bulls ready for sale means grain feeding. It will never be possible to have an animal fat without having a layer of fat in the scrotum, surrounding both testicles and the spermatic cord.Grain feeding in small pens means the bull will eat for a very short period of time and laydown most of the day. This inhibition of testicular cooling (no air flow) must, in my opinion, greatly exacerbate the fat testicle problem. In my opinion one thing we should be trying to do when feeding bulls is to arrange it so that they are forced to stand or walk for a good part of the day.On top of this, Bos Indicus bulls are bred to handle heat therefore they don’t even seek shade when lying down. At least a British bred bull would get in the dam or under a tree in the heat. Not the Bos Indicus, he just lays there making testicular jerky.
2. If the body is running a fever the blood pouring into the testicles will be a lot hotter than normal and the cooling mechanisms of the pampiniform plexus and scrotal muscles will be unable to cope. The detrimental effects of this depend on how much the temperature increases and the length of time it stays up.
3. When the ambient temperature causes the animal heat stress, the testicular temperature will rise just like the rest of the body.
4. Those bulls that have a short scrotum and are incapable of letting the testicles down, meaning those with a “wedge shaped” scrotum, struggle to get adequate thermoregulation. Think tight jocks.

2. STRESS

Regardless of its origin, stress lowers the testosterone levels. Remember that the epididymis relies on a very high level of testosterone to work well and that the final stages of sperm maturation occur here. So this is why a lot of DMR’s appear very soon (often within a week) after a stress.

Just as in humans, bulls are stressed by a long list of factors. Among these would be handing, dominance, trucking, nutrition and infection.

And just like humans, I feel (with no proof), that individual bulls handle these stresses differently. It is conceivable, therefore, that the same stress could not affect one bull at all, have a short term effect on another, and have a long lasting detrimental effect on another.

If you think about the detrimental effects listed, putting a bull through a sale will tick most of the boxes. In fact all bull sales including multi-vendor sales, private on property sales and out of the paddock sales will have a lot of these effects. So it would be surprising if the semen quality of a purchased bull did not decrease dramatically from his pre-sale tests if the bull is re-tested shortly after arriving home. A lot of bulls coming from such sales to Rocky Repro for example, will give freezeable quality semen in the first week to maybe two after arriving and then deteriorate for some time before coming good again (hopefully).

This is not because they were not fertile before they left home but because of what they have been through since leaving home.

Figure 10 illustrates how the stress of a mild illness can affect semen quality. This bull was noticed just to be “off-feed” and a little “doughy” on the 18/5/15, ruminations were slow and dung was firm, there was a very mild increase in temperature (39.5°C), otherwise no clinical signs of note. He was treated with antibiotics “just in case” and came back on feed within five days. Had this bull been grazing in the paddock no one would have noticed a thing wrong with him.

His morphology was terrible for two months following this upset before coming back to normal.

This case also demonstrates very well the 60-70 days it takes for semen production to return to normal after the testicular heeling has occurred. This is a classic example, but many bulls do not read the text book and take a lot longer.

3. PHYSICAL DAMAGE

This would include the following:

1. Self-inflicted;
2. Fighting; and
3. Accidental.

Most of these are self-explanatory, but a common case of self-inflicted damage is having a very long neck of the scrotum, whereby the testicles hang between the bulls hocks.

SUMMARY

1. Predicting a bulls life time fertility or even his fertility for the next 12 months based on a single test is always going to be very difficult given this is a biological system and fertility is a multi, multi-faceted and complex system.
2. There are infertile bulls out there that need to be identified.
3. Our current semen testing methods are good at telling the fertility of an ejaculate. AND,
4. Probably do a reasonable job of identifying the real losers. BUT,
5. Do a poor job of grading those that are acceptable. (are those that are OK versus good versus great any different – probably not).
6. Pre-sale semen testing results will almost always be better than the same tests run in the short term (one to two months) post sale.
7. The pre-sale tests simply say the bull was fertile at that time but his future fertility will vary depending on the degree of stress he incurs between the test and mating, and how the individual copes with that stress.

FIGURE 10: batch morphology

Images provided by Rocky Repro