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Proceedings of the 16th

Italian Association of Equine Veterinarians
Congress

Carrara, Italy
January 29-31, 2010

Next SIVE Meeting:

Feb. 4-6, 2011 – Montesilvano, Pescara, Italy

Reprinted in the IVIS website with the permission of the

Italian Association of Equine Veterinarians – SIVE

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Morphologic abnormalities
Abnormalities of
of sperm
Sperm

Claire Card
DVM PhD diplomate ACT, Dept LACS, Western College of Veterinary Medicine,
University of Saskatchewan, Saskatoon, Sk S7N 5B4 Canada
Claire.Card@usask.ca

Introduction: Van Leeuwenhoek in 1677 was semen1,2. The variation in the percentage of
the first scientist to observe sperm. His suc- normal spermatozoa among stallion seasons
cess was due to improvements in the quality accounted for 15% of the variation in stallion
of lenses that were available for microscopy. fertility, and the combination of sperm mor-
He observed what he described as “animalac- phology and motility accounted for 37% of
ula” in seminal fluid in a variety of species. the variation1. A fuller picture of a stallion’s
He noted and recorded the heterogeneity in fertility is achieved through a morphologic
sperm head shapes in men and other animal assessment of the sperm. A morphologic as-
species. Over time it was shown that beyond sessment of the sperm from 2 ejaculates is a
normal species specific differences in sperm, requirement for a Breeding Soundness Ex-
there were other morphologic changes present amination of a stallion. In addition in combi-
that were associated with abnormal sperm. nation with motility measurements and preg-
Many of these morphologic abnormalities of nancy rates, morphologic assessment allows
sperm were associated with either intrinsic or the detection of negative or positive trends in
transient male sub- or infertility. Many inves- spermatogenesis.
tigators usd systematic studies of sperm mor- The advent of cooled transported and frozen
phology to devise a number of classification semen has increased the number of semen
systems with the goal of finding an efficient samples handled by veterinarians. There are
and accurate way to evaluate the potential fer- currently few guidelines about semen quali-
tility of a male. ty, and many samples arrive with no infor-
mation.
Reason for Morphologic Assessment: Many A quick assessment of the sample allows for
stallions are investigated for their fertility po- a determination of the number of progres-
tential before retirement to stud, as part of a sively motile and morphologically normal
pre-purchase examination for a sire prospect, sperm available in the inseminate. A clini-
or prior to semen shipment or cryopreserva- cian is then in a position to determine if 1 or
tion. Stallions may also present with a history 2 insemination dosages were received, and
of low fertility, or declining fertility. Semen will gain insight into the mare’s relative
parameters are monitored during a breeding chance of conceiving based on the number
season to allow management adjustments if of morphologically normal and progressive-
the quality of the sperm produced declines, or ly motile sperm.
the pregnancy rate suffers. It is essential that
all health, fertility and semen characteristics Anatomy of a Sperm: Sperm have a head
are used in context when interpreting a mor- covered (2/3 of the head) by an acrosome to
phologic assessment of the semen. the equatorial region, a midpiece, a principle
It is customary when working with equine piece and an end piece. The supporting 9 +2
semen to only evaluate the concentration and arrangement of microtubule doublets running
motility of the sperm. Progressive motility is through the mid, principle and end piece com-
correlated with fertility, as is the dose of the prise the axoneme.
2
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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Choice of Morphologic Classification Sys- fore both Major and Minor, Primary or Sec-
tem: If we look to history to understand the ondary defects may be present on the same
methods of morphologic classification of sperm. The testes also produce sperm with
sperm we find that Lagerlof, Milovanov, and more than one type of Major defect on a sin-
Blom wrote about methods of classifying gle sperm. An example of this would be a
sperm in bulls. Blom described 2 systems of sperm with a large (macrocephalic) head and
classification where sperm defects were either broken midpiece. There are many situations
termed as “Primary or Secondary” or “Ma- where a sperm has more than one defect, these
jor or Minor”3, 4. In the first system 2 mor- Major Minor or Primary Secondary systems
phological categories for sperm defects were suggest that the most proximal defect or the
used. Primary defects were those that oc- most severe defect when classifying sperm
curred during spermatogenesis, thus repre- should be chosen. In the Primary and Second-
senting a failure of spermatogenesis; and Sec- ary defect system if a sperm has 3 defects such
ondary defects were those that occurred in the as nuclear vacuoles, a swollen midpiece and a
passage of the sperm through the excurrent proximal droplet, only a primary defect would
ducts, which represented a failure of matura- be enumerated, even though there is another
tion. A Primary defect was therefore deemed primary defect (the swollen midpiece), and
to be of testicular origin (examples include another secondary defect (proximal droplet).
nuclear vacuoles or acrosome defects), and a Using the Major Minor system where a sperm
Secondary defect would be a defect of matu- has a microcephalic head, a distal midpiece
ration such as a proximal droplet5. Some au- reflex, and a coiled principle piece with a dis-
thors referred to Tertiary defects, which are tal droplet, only one major head defect would
defects that occur from improper handling of be enumerated. The process of choosing a de-
the sample or faculty preparation of the slide. fect on a sperm, even when multiple defects
Blom’s classification system is one of the are present, results in a percentage distribution
most widely used system of classification of that will total to 100%. The examiner will not
sperm defects. Another classification system however have a record of the distribution of
by Blom used “Major or Minor” to describe the sperm defects in the ejaculate. This fact
defects4. Major defects included problems makes it much harder to determine if there are
with heads, midpieces, and proximal droplets, any changes in the types of defects present in
which are defects that are thought to have a sperm over time. This is relevant when we
greater impact on fertility. Minor defects were consider the process of spermatogenesis and
deemed those defects which have an unknown determine that multiple developmental stages
role or inconsequential role in fertility, such as of the sperm may be affected by an injury.
distal droplets. More recently a concept of classifying sperm
These are binary classification systems. There defects as Compensable and Non-compen-
are problems with using a binary process of sable System was also proposed6. A Compen-
classifying sperm. Using Blom’s systems sable defect is a defect where either the sperm
sperm are first classified as normal or defec- are not able to reach the site of fertilization, or
tive, followed by another binary classification the sperm is unable to: bind to the oocyte, in-
of the defects as Primary or Secondary, or in duce the cortical reaction, and initiate fertil-
the alternative as Major or Minor defects. The ization. There is no effect on fertility if a nu-
binary system means that each sperm must be merically adequate population of normal
assigned one category. The reality is that dur- sperm are present for fertilization, because the
ing spermatogenesis billions of sperm are pro- oocyte may still be fertilized by other fertile
duced, and inevitably some abnormal sperm sperm. The fertility of the individual is not af-
are produced. Testis tissue produces sperm fected by defects that are effectively overcome
with both Major and Minor defects, and sperm by having adequate levels of normal sperm.
with a Primary defect(s) may gain a Second- The normal sperm compensate for the abnor-
ary defects as they undergo maturation. There- mal sperm. Similarly a non-compensable de-
3
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fect is a defect that does not interfere with the Preparation of a sample: In a complete se-
sperm reaching the site of fertilization, and the men analysis the total sperm number is deter-
cortical reaction is induced by the sperm as it mined (concentration x volume), the motility,
binds to the oocyte, and may cause syngamy, and the morphology of the semen is exam-
but the sperm is defective in that it is capable ined. The stallion’s sperm sample must be fil-
of supporting embryonic development. These tered to remove the gel fraction because gel
non-compensable sperm defects interfere with makes the sample impossible to interpret.
fertility. An example of a non-compensable Sperm are mixed with Eosin Nigrosin for the
defect is sperm DNA condensation or DNA morphologic assessment A 60:40 stain to
compaction problems. sperm ratio is recommended to provide good
contrast and density of sperm for evaluation.
Introduction to the Differential Spermiogram: The stain is hyposmotic to the sperm so the
Another popular system of morphologic evalu- slide should be dried quickly, such as on a
ation is called the Differential Spermiogram7. warming tray, to prevent any hyposmotic arte-
The Differential Spermiogram System is per- facts from forming (usually curved mid and
formed in an analogous fashion to a differential end pieces). Wet mount preparations viewed
white count in a leukogram. A total white count under phase contrast do not usually allow the
and a differential cell count is performed on a same detailed examination of the defects pres-
blood sample. The differential white blood cell ent. The examination is performed using 100x
count gives us a variety of information such as objective (oil immersion) which yields 1000x
the percentage of lymphocytes, neutrophils, magnification7.
monocytes, eosinophils, and basophils in a sam-
ple. Changes in the absolute number and per- Laboratory Errors in Preparation: Labora-
centages of neutrophils and lymphocytes are tory errors causing the appearance of sperm
evaluated because they provide us with valu- defects are sometimes referred to as tertiary
able clinical information. Neutrophils are also defects. There are a few defects that may be
scored for signs of activation, such as toxic produced through errors in sample handling
changes, which indicate impending cell death. such as failure to filter the semen, excessive
Serial daily and weekly samples are used to stain, cold shock and hyposmotic shock. Gel
compare changes in terms of the absolute cell in the sample makes the background appear
numbers, percentages of cells such as neu- white and makes the sperm difficult to see.
trophils, and the condition of the cells in terms Excessive stain will create stain cracks along
of toxic changes in the leukogram. This infor- the sperm. Artefacts associated with cold
mation helps us to determine if our patient is shock of sperm include distal midpiece reflex-
improving, staying the same or deteriorating. es, while hyposmotic shock may result in
This same principle applies to use of the Differ- curved midpieces or curled endpieces. In gen-
ential Spermiogram in the stallion. The slide is eral it is very important to emphasize that the
examined for the different cell types in the vast majority of defects present in a sample
ejaculate (sperm, neutrophils, lymphocytes, come out of the stallion. Errors or rough han-
red cells, squames, germinal epithelial cells). dling of the sample will not produce defects
The sperm cells are differentially classified such as detached heads, or nuclear vacuoles.
by enumerating all the defects on the sperm. Most sperm defects are either developmental
Using the supravital stain Eosin Nigrosin, live or maturational in nature.
and dead sperm are evaluated in a separate
count. Cells which stain pink are considered Differential spermiogram: A spermiogram,
dead or devitalized, while the cells that stain which is a frequency distribution of all de-
white are considered alive. The Live / Dead fects is performed on a minmum of 100
sperm ratios are determined, to indicate the sperm cells, using a cell counter, and a sepa-
vitality of the sperm, similar to the identification rate live dead count of at least 100 sperm are
of toxic changes in neutrophils. performed8. All the sperm in a representative
4
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS

microscopic field are categorized. The pres-

ence of other cell types is noted, and addi-
tional stained slides may be determined to
identify the other cell types. Therefore if a
sperm has a macrocephalic head and a mid-
piece defect both are enumerated. To enumer-
ate both defects simultaneously one presses
both keys down at the same time on a cell
counter device. This will only advance the
counter by one number in the total column.
For example using a cell counter if 100 sperm
are counted and all the abnormalities are enu-
merated (meaning one presses more than one
key at the same time), only one sperm is
added to the total count even when more than
one defect is present on a sperm. Mathemati-
cally the percentages for the categories of de-
fects when added to the percentage of normal Figure 1 - Normal sperm.
sperm will not add to 100% using the Differ-
ential Spermiogram System.
Subcategories of sperm: There are also sub-
Categories in the Differential Spermiogram: categories of the head and midpiece defects,
The categories used in the Differential and the relative frequency of occurrence with
Spermiogram include: head, midpiece, de- the most common subcategory being noted
tached normal head, detached abnormal first, followed by the others in a descending
head, principle piece, proximal droplet, fashion. This is done by memory. The subcat-
distal droplet, and acrosomes. Germinal egories of the main defects include:
epithelial cells are counted if more than just
an occasional cell is present. All the sperm in Head defects: microcephalic (too small),
a field are counted irrespective of whether macrocephalic (too large), pyriform (pear
they stain white or pink with the Eosin Ni- shaped), tapered (long and narrow sperm)
grosin stain. Figure 1 shows a normal sperm. teratoid (almost non-recognizable) and vac-
When detached heads are encountered the uolated. There are a number of reports on the
head but not the corresponding headless mid- head morphometry of sperm in a large number
pieces are counted7. of breeds10-12. The range of values for fertile
This will only advance the counter by one stallions was as follows: length, 4.9-5.7 µm;
number in the total column. For example us- width, 2.5-3.0 µm. Higher percentages of
ing a cell counter if 100 sperm are counted morphometrically normal sperm heads are
and all the abnormalities are enumerated found in fertile than in subfertile stallions (52
(meaning one presses more than one key at vs. 19%)11. Essentially sperm are twice as
the same time), only one sperm is added to long as they are wide, and variations in size
the total count even when more than one de- resulting in sperm heads that are >25% larger
fect is present on a sperm. Mathematically the or smaller should be considered abnormal.
percentages for the categories of defects Stallions however are noted as very having a
when added to the percentage of normal variety of head shapes and sizes in their ejac-
sperm will not add to 100% using the Differ- ulates. The exception is in the Warmblood
ential Spermiogram System. Certain abnor- breeds where selection for fertility has result-
malities when found together represent more ed in a much more uniform population of
severe disturbances in spermatogenesis and sperm with consistent head shapes and sizes.
influence the stallion’s prognosis8,9. Pyriform sperm (pear shaped with a narrow
5
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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base, and a proportionately wider apex) may

be associated with chromatin instability. Ta-
pered cells are long and narrow and are may
not be significant if the whole population of
sperm are uniformly tapered. Teratoid sperm
have the mid and principle coiled around
head. Vacuoles in sperm are visible as dark
spots on the sperm head when using. Small
single apical vacuoles may not be highly sig-
nificant in stallions. Larger and more conflu-
ent vacuoles are of significance. Eosin Ni-
grosin. The overall frequency of the head de-
fects present in both the intact and detached
sperm are noted.
These are listed from most frequent to least
frequent. Regarding acrosomes, knobbed,
missing, or abnormal acrosomes are counted
separately. Figures 2 and 4 show sperm with Figure 2 - Sperm with pyriform head/segmen-
pyriform, vacuolated heads, and a knobbed tal aplasia of the mitochondrial sheath (upper)
acrosome. and vacuole/pyriform head/segmental aplasia/
abnormal principle piece (lower).
Midpiece defects: The subcategories of the
midpiece defects include: segmental apla-
sia of the mitochondrial sheath, swollen
mitochondrial sheath, pseudo-droplet,
fractured midpiece, and distal midpiece
reflexes. Abaxial insertion of the midpiece
is not considered an abnormality in the
stallion or the boar. Figure 3 shows sperm
with fractured midpieces. Segemental aplasia
of the mitochondrial sheath has the appear-
ance of gaps in the mitochondrial shealth, a
swollen sheath involves visible thickening of
the sheath, a pseudo-droplet is bunching of
the mitochondria on the midpiece with a cor-
responding region on the midpiece of miss-
ing mitochondria, fractured midpieces usual- Figure 3 - Sperm with broken midpieces (all)
ly have broken fibres extruding at the site of and proximal droplets (left and uppermost).
the break, a distal midpiece reflex involves a
hairpin turn of the principle piece with or
without a distal droplet. Proximal droplets
are made of cytoplasmic material and are defects are present on a sperm it is not as-
considered separately and are not a midpiece sumed that one defect is more important than
defect per se. another. The examiner is enabled to determine
the potential of the defects to interfere with
Summary: The Differential Spermiogram fertility.
System allows investigation of changes in the The enumeration all of the defects for each
frequency or the prevalence of the defects sperm allows the chronological pattern of the
recorded over time. All the sperm in a repre- changes over time to be considered (increase,
sentative field are categorized. When multiple decrease, stay the same).
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Comparing the Differential Spermiogram

to Other Classification Systems: The per-
centage of normal sperm is a constant in all of
the classification systems. Therefore this
measure should be same in all systems.

Review of Spermatogenesis in the Stallion:

During spermatogenesis diploid germinal
cells termed spermatogonia, replicate and ma-
ture to form haploid, mature sperm. A stallion
produces an estimated 16 million sperm per
gram of testicular tissue per day. The defective
sperm are eliminated through apoptosis and Figure 4 - Sperm with a loose head (left),
phagocytosis by the Sertoli cells, and other pyriform head/vacuole/distal midpiece reflex
phagocytic cells in the excurrent ducts, while (centre) and a knobbed acrosome/pyriform
others are passed into the ejaculate. The sem- head (right).
iniferous tubules contain the germinal or sper-
matogenic epithelium and the supportive Ser-
toli cells. Cross sections of the seminiferous genetic material into haploid spermatids (19.4
tubules show that 4 or 5 specific cell types are days), and spermiogenesis development and
consistently found together, and these are differentiation (18.6 days). Spermiation is the
present in different combinations called cellu- release of spermatids into the lumen of semi-
lar associations. The successive progression in niferous tubule. Approximately 9 days are re-
the cellular associations requires 12.2 days in quired for transportation of sperm through the
the stallion. The repetitions of the 12.2 day pe- excurrent ducts such as the rete testis, and epi-
riod means there is a progression of the cells didymides. A new population of sperm are
through 8 stages which are numbered I available for ejaculation every 64-66 days. In
through VIII. The time frame to pass though the absence of ejaculation sperm are eliminat-
each stage is not equal. The stages can be dif- ed in the urine. There is no seasonal influence
ferentiated by a detailed examination of the on the developmental intervals, in other words
spermatocytes, spermatogonia, and sper- in winter or summer a sperm takes the same
matids in a cross section. The succession is amount of time to produce sperm. There is a
evident when the viewing the rings of the ger- seasonal influence and an influence of age on
minal cells in the tubule. The cells located in total sperm production, sperm motility and
the outer ring of the tubule replicate so that sperm morphology.
over time more differentiated cells are formed,
which through successive generations, results Stallion sperm morphology: A number of
in the most differentiated cells being located authors have reported that the percentage of
closer to the lumen13. morphologically normal sperm in the general
population of stallions is around 50%14,15.
Time Frame: The time period from a com- Therefore in fertile horses there are a relative-
mitted spermatogonium to a mature to a ly large number of morphologically defective
sperm that is released in the lumen of the sem- sperm9. This is important to remember, but
iniferous tubule has been shown experimen- pasture mated and feral horses achieve high
tally to be around 55 - 57 days in the stallion. fertility rates under good nutritional condi-
This 55 - 57 day period may be divided into 3 tions16. The question then becomes determin-
phases: spermatocytogenesis where mitosis ing what constitutes a problem when the per-
and differentiation of spematogonia occur centage of morphologically normal sperm is
(19.4 days); meiosis the period of replication lower than 50%, and / or mares are failing to
(primary spermatocytes) and then reduction of conceive. A high percentage of defects is as-
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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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sociated with lower fertility17,18. Selection for ment of patent seminiferous tubules. This ex-
fertility such as in Warmblood breeds does re- plains why germinal epithelial cells appear in
sult in higher percentages of normal sperm19, the ejaculate at this time. The process of sper-
or in the case of inbred breeds such as: matogenesis is not highly efficient when pro-
Clydesdales, Shires, Tenneesee Walkers, and duction first begins and there are increased
Friesians lower fertility and poorer sperm numbers of abnormal sperm. Four types of
quality is present20. defects are common in pubertal stallions; ger-
minal cells, head defects, midpiece defects,
Serial evaluation of the Sperm Morpholo- and proximal droplets. A total scrotal width
gy: Serial examinations of semen using the of 8 cm and soft testes usually indicates that
Differential Spermiogram system, is a power- the stallion is immature. A typical ejaculate of
ful clinical tool when it is possible to gain in- a pubertal stallion has a low concentration,
sight into the fertility potential of an individ- low motility, high percentage of germinal
ual. The problems that arise as a result of the cells, and other defects such as head, mid-
stallion’s intrinsic fertility (genetic), a point piece and proximal droplets. The second ejac-
source problem such as a stress (fever), or a ulate shows similar parameters except the
long standing alteration (testicular degenera- concentration plummets revealing a low re-
tion) may be diagnosed by serial examinations serve capacity.
of ejaculates spaced at least 1 month apart. The A pubertal stallion should be classified as
clinical interpretation of semen samples is Questionable Breeding Prospect, and re-eval-
challenging because an examiner must deter- uated in 2-4 months time or longer depending
mine which parameters and abnormalities are on the photoperiod conditions. A recheck ex-
constitutive and reflect an individual stallion’s amination should show the following an in-
age and intrinsic genetic ability, and which crease in: total sperm numbers, sperm con-
changes are extrinsic due to a disturbance (nu- centration, motility and percentage of mor-
tritional, hormonal, infectious, toxic, degener- phologically normal sperm with a decrease in
ative, idiopathic) in spermatogenesis21. germinal epithelial cells, proximal droplets
and head/midpiece defects. If the stallion has
Age related changes: In general stallions poor intrinsic fertility he will not show signif-
reach puberty at about 18 months of age, but icant improvement over time. It is important
there is wide variability with stallions as to determine if the stallions may be under
young as 8 months achieve puberty. Testicular stress or is receiving anabolic steroids or glu-
size is correlated with fertility22. Larger ma- cocorticoids which may negatively influence
ture size breeds tend to mature later. Mature sperm morphology.
stallions will have an increase in testicular
mass until around 4 years of age. Older stal- Intrinsic Defects: In a population of male an-
lions tend to be more seasonal than younger imals examined for fertility there will be: be-
stallions, but in general fertility does not de- low average, average and above average levels
cline with age in horses23,24. of fertility, which are reflected in their testicu-
lar size, intrinsic sperm number/concentration
Puberty - Young stallions at 2 years of age and motility / morphology parameters. Intrin-
may still be exhibiting signs of puberty, while sic defects are consistent and there will be on-
others may have poor intrinsic fertility. Until ly minor seasonal fluctuations, and therefore
a stallion is producing at least 100 million there is no improvement over time. The abili-
sperm per ejaculate he is not considered to ty to produce sperm is determined by the stal-
have reached puberty. Stallions reaching 18 lion’s genetics and testicular size. The consis-
months of age in the winter may have a de- tent appearance of a high percentage of mor-
layed onset of puberty related to photoperiod. phologically defective sperm in an otherwise
The process of puberty takes approximately 2 healthy young stallion who has no history of
months to complete and involves the attain- illness and who is not receiving exogenous
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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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hormone therapy suggests that he has intrinsi- number of sperm, progressively motility of the
cally low fertility, and he will exhibit only mi- sperm, and the features of the Differential
nor seasonal variations in his semen. The pres- Spermiogram the status of the stallion’s fertil-
ence of intrinsically low fertility is accentuat- ity and progression through the breeding sea-
ed by inbreeding, and is supported by the son may be followed. During the examination
practice of not selecting for fertility character- of a stallion the morphology of the sperm
istics in stallions. In some stallions these are must be integrated with the rest of the infor-
manifested by consistent defects present in mation about the patient. This includes the
high percentage of sperm, visible at the light historical information on per cycle pregnancy
and ultrastructural level, such as Dagg-like rates, early pregnancy losses, the testicular
sperm (fractured midpieces), or missing size / consistency, and the features of the ejac-
dyneine arms (immotile live sperm)25,26. ulates such as total sperm number, concentra-
tion and volume, percentage of progressively
Disturbances in Spermatogenesis: A distur- motile sperm and percentage of morphologi-
bance in spermatogenesis is generally mani- cally normal sperm.
fested by changes in the sperm morphology as
quantified using a Differential Spermiogram. Temporary fertility impairment: Temporary
Insults to spermatogenesis may be transient fertility impairment may arise from a point
and result in temporary fertility impairment, source insult. These types of injuries represent
or may be long standing and result in perma- Extrinsic influences on spermatogenesis. Typi-
nent fertility impairment. Temporary insults to cally a change is noted over time or historical-
spermatogenesis for example may result from ly fertility was acceptable in the recent past28.
fever, stress, exposure to toxins, trauma, nutri- Examples of point source insults include tran-
tional deficiencies/toxicities. Damage to the sient high fever or scrotal heat stress29,30. Seri-
progenitor spermatogonia, which are required al evaluations of multiple ejaculates and an
to replicate in order to replenish the popula- examination of the changes in the proportions
tion of dividing cells, may be severe enough to or prevalence of the various sperm morpho-
result in a permanent reduction or loss of logic defects provide the best information on
sperm production. the nature of and subsequent recovery from a
In general the age of the stallion, season of the disturbance in spermatogenesis7. A point
year, nutrition, health status, medications, and source insult creates damage to the cells pres-
the general stress the horse is under impact ent at the time, generally producing a variety
sperm morphology27. Adverse exposure to tox- of head and midpiece defects. Due to the
ins, radiation, dietary deficiencies or excesses, length of the time required for spermatogene-
heat stress, trauma, parasite migrations, tu- sis at least 64 days plus the time for transit
mours etc also influence sperm morphology. A through the excurrent ducts must elapse be-
disturbance in spermatogenesis is therefore su- fore a new population of cells is ejaculated.
perimposed on the other factors that influence Sperm that are injured that are close to matu-
sperm production. The features of the sperm in rity appear first in the ejaculate, while sperm
the ejaculate provide a snapshot of the cumu- that are immature would appear later. This has
lative health of the seminiferous epithelium, been shown by studies that examined semen
sertoli cells and epididymides. The intensity, characteristics after steroid administration
nature, duration, and sensitivity of the sperm (dexamethasone) or thermal insults (scrotal
cell types to an insult will determine the im- insulation)31,32. Changes noted in sperm mor-
pact on the Differential Spermiogram. Note phology after scrotal insulation began to ap-
that all of the sperm cell types, stages, and cel- pear 10 days after insulation, and then peaked
lular associations are present in all stallions. at about 30 days. Preinsulation levels of nor-
By integrating the information on history, age, mal sperm were not reached until 50-75 days
season, environment, nutrition, ejaculatory has elapsed after insulation32. During a serial
characteristics, such as concentration, total examination of sperm morphology if the per-
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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS

centage of normal sperm increases and the

other categories of defects decrease, this
would indicate a recovery from the problem,
but a lack of change or a decline in the per-
centage of normal sperm indicates an ongoing
problem. The presence of specific cellular as-
sociations in the stallion means that certain
cell types may be damaged at the same time.

Stress - Athletic horses are subject to a num-

ber of stressors such as high environmental
temperatures, transport stress, chronic pain
due to lameness, and medications such as an-
abolic steroids. Generally this type of stress is Figure 5A
manifested as a decline in the percentage of
motile and normal sperm, which is reversible
if the stress is removed.

Permanent Fertility Disturbances: A vari-

ety of problems may result in permanent fer-
tility impairment such as tumours of the testis,
testicular torsion/infarction, heavy metal ex-
posure, or unilateral castration. Severe nutri-
tional, hormonal, traumatic, infectious, toxic,
or degenerative insults may permanently im-
pair fertility. These conditions may alter scro-
tal thermoregulation, cause intense inflamma-
tion, may breach the blood testis barrier, inter-
fere with endocrine function, or damage the
Figure 5B
spermatogonial progenitor cells. More often
than not in stallions the cause for the decline
Figure 5 - Top panel A shows an Eosin Ni-
in fertility is not identified and is therefore id-
grosin stain of an ejaculate, the dark round
iopathic. The most common cause of idio-
cells are germinal epithelial cells. The bottom
pathic sub or infertility in stallions is testicu-
panel B is a Diff Quik stain of the same sam-
lar degeneration. Occasionally testicular de-
ple, demonstrating that the cells are not neu-
generation is transient following a dramatic
trophils.
insult, but in most cases it results in perma-
nent alterations.

Testicular Degeneration: The history of the (ribs)33. A Differential Spermiogram will

stallion generally includes that previously ac- show low numbers of normal sperm, many
ceptable (45-55%) per cycle pregnancy rates germinal epithelial cells (>10%), and high
has declined to a lower per cycle pregnancy percentages of abnormal sperm. Head and
rate (<30%). This history should trigger a full midpiece defects may exceed 25-30% see
breeding soundness examination including an figure 5. The potential for improvement is low
evaluation of the stallion’s testicular size and based on testicular consistency and size. The
consistency. Testicular degeneration results in poor motility of the sperm often relate to un-
a loss of germinal epithelial cells, and will re- derlying morphologic problems of the mid-
sult in small testes, with a soft consistency piece. An excess number of germinal epithe-
with prominent connective tissue bands lial cells, high percentages of head and mid-
10
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS

piece defects should suggest testicular degen- specific order and in a specific time frame. We
eration. Serial examinations will show persist- know that immature sperm, such as those with
ence of the problem or a continued decline. proximal droplets, are not able to fertilize be-
These stallions would be classified as having cause their DNA is not fully condensed. The
an Unsatisfactory Breeding Potential. The droplet present on sperm begins is in a proxi-
stallions with testicular degeneration will tend mal location on the midpiece as the sperm en-
to stay the same or decline in the percentage ters the epididymis, but is shed from the distal
of normal sperm over time. True testicular de- piece just prior to ejaculation. Proximal
generation is not reversible, and improved fer- droplets in a high percentage of sperm indicate
tility relies predominately on improving the that these sperm are immature and have not
management of the stallion. completed chromatin compaction.
The formation of the acrosome occurs concur-
Subfertility: A subfertile stallion may be in- rently with some stages of chromatin com-
trinsically an inefficient breeder, or he may paction. The presence of acrosomal defects
have lost fertility over time. Therefore the his- suggests that additional tests should be con-
tory is very important is determining the na- sidered (chromatin assays), as the sperm with
ture of a stallion’s subfertility. Stallions loose knobbed acrosomes have been shown to have
commercial viability when their per cycle more immature chromatin38. Sperm with acro-
pregnancy rates drop below 30%. There are a somal defects are a heterogenous population
host of reasons why stallions become subfer- and morphologic problems such as mild knob-
tile, including psychological and medical bing or folding of the acrosome may not in-
problems. However the majority of subfertili- fluence fertility, but extremely severely
ty problems are manifested by high percent- knobbed acrosomes or those that contain
ages of morphologically abnormal sperm, and vesicular material represent a more serious
the stallion may or may not have low sperm form of the defect, and are more likely to have
motility. The presence of germinal epithelial an underlying problem in chromatin matura-
cells indicates the stallion may have testicular tion. The knobbed acrosome defect may have
degeneration, but the absence of germinal ep- a heritable basis, and may be a constitutive
ithelial cells indicates the stallion has poor problem in some stallions. Therefore one ab-
sperm quality. Ultrastructural defects may be normality may signal the presence of another,
present in the sperm. Problems with the head, as acrosome defects are associated with chro-
annulus, midpiece and axoneme have been re- matin defects. Serial semen evaluations may
ported25,34-37. be used to determine the persistence of the de-
fect39. There is some suggestion that this con-
Defects: For example severely knobbed acro- dition is heritable in many species40.
somes, midpiece defects, DNA condensation
defects, and nuclear vacuoles may be present Spermiostasis: Spermiostasis is usually an
in sperm that are alive and progressively acquired and persistent idiopathic condition in
motile in stallions with a testicular size/mass the stallion. In these stallions one or both am-
and consistency within normal limits. Sper- pullae fail to function normally and sperm ac-
matogenesis is a complex process, which in- cumulate. The ampullae may be partially ob-
cludes dramatic changes in the chromatin, structed or completely blocked. In the case of
which refers to DNA and the complex of pro- complete obstruction alkaline phosphatase
teins that associate with it. During maturation will still usually be elevated in the semen sam-
in the testes and epididymides there are a num- ple indicating the horse is ejaculating. The
ber of protein substitutions, often with zinc typical spermiogram shows extremely high
containing proteins, that neutralize the charges numbers of sperm with detached usually pink
on the DNA, and allowing it to compact or fold staining heads, either in every ejaculate or re-
up. This process is known as DNA condensa- leased intermittently. The ampullary plugs are
tion. The proteins need to be substituted in a occasionally released and grossly visible
11
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS

structural analysis12,39, such as with immotile

sperm that stain alive (primary ciliary dyski-
nesia)25,34,36,37,40,41. Occasionally testicular
biopsy is used to determine if the appropriate
cell types are being produced and to confirm
where the defective sperm are forming, or if a
tumour is present42-44.

Treatments to improve sperm morphology:

There continues to be interest in improving se-
men quality through the use of supplements
such as docosahexaenoic acid (DHA), and
omega 3,6 containing neutriceuticals. Others
Figure 6 - Abnormal sperm in a stallion with suggest supplements such as carnitine, and vi-
spermiostasis, note the presence of many de- tamin E selenium may be beneficial. The
tached heads. DHA products are derived from fish meal and
are fed for months before a desired effect is
reached. Some reports show improvements in
within the ejaculate. The detached heads are sperm morphology, motion characteristics,
believed to belong to senescent sperm that are and ability to survive cooling and freezing45,46.
then improperly stored and intermittently re- Many times efforts are aimed at enriching the
leased. Cannulation and flushing of the am- stallion’s environment psychologically so he
pullae through a urethral approach has not achieves harem status, and are focused on
been successful. The underlying pathogenesis medical management to remove stress, im-
of the condition remains obscure. Rectal mas- prove health and ensure the stallion is fully
sage of the ampulla, and oxytocin before col- ejaculating.
lection have been used along with frequent col-
lection to manage some stallions (Figure 6). Adjustments to Breeding Doses Manage-
ment Routines: Stallions with high percent-
Supplemental tests: When sperm appear to ages of abnormal sperm may still successfully
have normal morphology but do not get mares get mares in foals at acceptable rates if they
in foal it is time for a different level of exam- have enough vigorous normal sperm to com-
ination13,33. Transrectal ultrasound and testicu- pensate, especially when breeding at pasture.
lar ultrasound to evaluate testicular echotex- Planned multiple mating have been reported
ture, and Doppler analysis of blood flow may to increase pregnancy rates in some stallions47.
be used. A variety of stains such as coomasie Insemination with whole ejaculates in stal-
blue, or fluorescent stains may be used to lions with very low numbers of normal sperm
evaluate the acrosome, and tests to provoke has also been used as a strategy to increase
the acrosome reaction may be performed. En- pregnancy rates25. Therefore when defects are
docrine tests are used to characterize the prob- compensable increasing the number of sperm
lem and responsiveness of the hypothalamo- in an ejaculate will often achieve the desired
pituitary-testis axis. A Feulgen stain may help result. Sperm samples with high percentages
identify nuclear vacuoles and chromatin con- of abnormal sperm on the average do not cool
densation defects. A sperm chromatin struc- as well or freeze very well. Veterinarians may
ture assay may be used to investigate chro- need to adjust the number of sperm in a breed-
matin stability. An antisperm antibody assay ing dose upwards to compensate for poorer
may be indicated if sperm are observed to ex- morphology. On the average stallions produce
hibit head to head agglutination. Scanning or around 50% normal sperm, a recommendation
Tranmission electron microscopy may show is adding at least 100 million more motile
or reveal the nature of a defect through ultra- sperm for every 10% decrease below 50%
12
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS

in morphologically normal sperm9. Stallions 8. Brito, L.F.C., Evaluation of stallion sperm mor-
generally will begin to leave unacceptably phology. Clinical Techniques in Equine Practice,
2007. 6(4): p. 249-264.
high numbers of mares open when they fall 9. Card, C., Cellular associations and the differential
below 100 million morphologically normal spermiogram: making sense of stallion spermato-
and progressively motile sperm per breeding zoal morphology. Theriogenology, 2005. 64(3): p.
dose. There are some papers that suggest that 558-567.
additional processing steps such as density 10. Hidalgo, M., et al., Morphometric classification of
Spanish thoroughbred stallion sperm heads. Animal
gradient centrifugation, glass wool sephadex Reproduction Science, 2008. 103(3/4): p. 374-378.
separation, or pelleting sperm followed by 11. Gravance, C.G., et al., Quantification of normal head
swim up may assist in harvesting the best morphometry of stallion spermatozoa. Journal of Re-
sperm from an ejaculate48,49. The percentage production and Fertility, 1996. 108(1): p. 41-46.
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lion semen using transmission electron microscopy.
with intact chromatin was increased by col- Animal Reproduction Science, 2006. 91(3/4): p.
loidal centrifugation. The authors reported a 285-298.
decrease in the incidence of proximal cyto- 13. Varner, D.D. and L. Johnson. From a sperm’s eye
plasmic droplets and midpiece defects after view - revisiting our perception of this intriguing
centrifugation through colloids. Whether fer- cell. in Proceedings of the 53rd Annual Convention
of the American Association of Equine Practition-
tility is improved by such processing steps ers, Orlando, Florida, USA, 1-5 December, 2007. p.
will vary with individual stallions, and likely 104-177.
would only apply to subfertile stallions, and of 14. Bielanski, W., et al., Some characteristics of com-
these stallions only a proportion of the them mon abnormal forms of spermatozoa in highly fer-
would realize an increase in fertility49,50. tile stallions. Journal of Reproduction and Fertility,
1982(32, Supplement): p. 21-26.
15. Bielanski, W. and F. Kaczmarski, Morphology of
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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010