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ABSTRACT
Assistant reproduction technologies are in constant evolution, among them the artificial insemi-
nation (AI). AI has been successfully used in pigs for decades, especially to improve boar effi-
ciency and productivity. Lately, swine AI has taken on a new lease of life since efficient AI is
essential for solving future challenges in the porcine industry and to enhance productivity. The
present paper summarizes several factors concerning AI, starting with an overview of some
physiological aspects including the female reproductive tract and sperm transport, as well as
sperm losses during insemination and uterus sperm selection. Strategies developed to reduce the
number of sperm during the AI process, are also reviewed, along with their combination with
new reproductive technologies for application in pig production in the near future.
KEYWORDS: swine, artificial insemination, spermatozoa, post-cervical, backflow, reproductive
technology.
An overview of swine artificial insemination: Retrospective,
current and prospective aspects
INTRODUCTION
In the last two decades assistant repro-
ductive technology (ART) has grown expo-
nentially due to the development of new bio-
technologies both in humans and animals. Ar-
tificial insemination (AI) is included among
ART methods. This technique, although not as
new as others, is still considered to be one of
the most revolutionary techniques applied in
farm animals. It can be defined as a method of
assisted reproduction that involves the deposi-
tion of sperm unnaturally in the female tract
for the purpose of fertilization. Although the
use of AI in most countries with intensive pig
production has increased greatly in the past
two decades, AI in swine cannot be consid-
ered a new technique. The first AI attempts
were recorded in the 14th century and its in-
troduction in porcine dates from the beginning
of 20th century (see section History of AI).
Nowadays, more than 90% of pigs are artifi-
cially inseminated in the European Union and
North America, reaching 98% in some coun-
tries (Feitsma, 2009).
This reproduction method presents
great advantages over natural mating. In this
respect, the following advantages can be em-
phasized: a genetic gain with the use of genet-
ically superior males and purchased semen al-
Journal of Experimental and Applied Animal Science
Volume 1, Number 1, pp. 67-97, 2013
Print ISSN 2314-5684 | Online ISSN 2314-5692
REVIEW ARTICLE
Soriano
-
Úbeda C,
Matás C and García
-
Vázquez FA
Department of Physiology, Faculty of Veterinary Science, University of Murcia, Campus Mare
Nostrum, 30100, Murcia, Spain
Soriano-Úbeda et al., 2013
Journal of Experimental and Applied Animal Sciences.
1, 1:67-97
68
lows genetic diversity, which can be used to
optimize crossbreeding systems on smaller
farms and increased genetic progress. Addi-
tionally, the number of boars can be reduced
on the farm since good males can be used
more extensively than those used for natural
service, because AI increases the number of
inseminations per ejaculate. Furthermore, this
technique presents less risk of disease trans-
mission than natural service mating systems.
However, AI requires a high level of man-
agement on the part of the farmer. The techni-
cian making the AI should provide special at-
tention to the handling of semen as regards
environmental changes during transport, in-
cluding temperature and the risk of dilution
that will affect viability. Also AI should be
carried out at the right time and the farmer
must make an accurate determination of the
onset of estrus. This fact is essential for ob-
taining a high rate of conception and litter size
(Maes et al., 2011).
The main goal of the sperm when they
are deposited in the female tract is to reach the
oocyte and fertilize it. Millions of sperm are
placed in the female, but only some ‘privi-
leged’ spermatozoa arrive at the fertilization
site (see section Sperm transport through the
female reproductive tract). During this long
journey through the uterus in search of the oo-
cyte, the sperm are subjected to different envi-
ronments and obstacles (see section Sperm
losses in the uterus during AI) so that only the
most ‘capable’ spermatozoa are chosen; how-
ever, the exact mechanism by which sperma-
tozoa are selected in the uterus is still not well
understood (see section Sperm selection in the
uterus after insemination). This information
leads one to think of genetic material wasted
on the journey and the inefficiency of the tra-
ditional AI technique. That is why in the last
decade new insemination devices have been
developed with the goal of reducing the num-
ber of sperm deposited placing them deeper in
the female tract and, as a consequence, closer
to site of fertilization (see section AI method-
ologies). Beside adopting new devices, the
porcine industry is trying to maximize sperm
use by the application of new methodologies,
such as improving the composition of liquid
storage, releasing the sperm progressively in
the uterus (encapsulated sperm) or including
new quality sperm assays which could permit
optimization of the ejaculate (see section New
reproductive technologies performing AI).
Although AI in swine is widely used,
its use is still below that present in bovine
species. Perhaps the reason is based on the
storage of semen fresh vs. frozen. In porcine,
over 85% of these artificial inseminations are
performed on the day of or on the day follow-
ing sperm collection using boar semen pre-
served at a temperature of 15-20º C. Although
frozen boar semen has been available com-
mercially since 1975, both in pellet form and
in straws, less than 1% of all inseminations are
made using frozen-thawed (FT) semen (John-
son et al., 2000) (see section Application of AI
to ‘special sperm’). Slowly swine AI is
spreading to other areas such as obtaining par-
ticular sex litters or genetically modified ani-
mals by the use of ‘transgenic sperm’ (see sec-
tion Application of AI to ‘special sperm’)
which, in future, could represent a tool for im-
proving the efficiency of meat production or
increasing disease resistance.
These and other aspects mentioned in
the introduction will be reviewed more deeply
in the following sections.
HISTORY OF AI
Although most people assume AI to be
a recent development, it was first used in the
14th century. The legend says that the first AI
successfully performed was in the equine spe-
cies, when an Arab chieftain stole ejaculated
semen from the vagina of a recently mated
mare belonging to a rival. The semen, theoret-
ically of better quality, was diluted in camel
milk and inseminated in the new mares (re-
viewed by Allen, 2005). Later, Leeuwenhoek
in 1678 using his own created microscope was
the first person to observe a sperm, something
that he called “animalcules” or “spermatick
worms” (Clarke, 2006). The following centu-
Soriano-Úbeda et al., 2013
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1, 1:67-97
69
ry, an Italian priest, Spallanzani (1784), exam-
ined semen from mammals, fish and amphibi-
ans and managed to perform the first success-
ful documented insemination in a dog, obtain-
ing three pups 62 days later. Moreover, exper-
imenting with frogs he demonstrated that pre-
vious contact between oocytes and spermato-
zoa is essential to obtain a tadpole. Perhaps,
this was the first experiment of in vitro fertili-
zation in the world. He also observed for the
first time that the spermatozoa could be inac-
tivated by cooling and reactivated later. Ap-
parently, as early as 1776 he put a sample of
collected human semen in the snow and dis-
covered that the spermatozoa were still motile
when they were returned to body temperature.
The first known human insemination was
made in 1790 by a Scottish surgeon, John
Hunter, who collected semen from a merchant
with hypospadias in a hot syringe. Following
the instructions from the doctor he successful-
ly injected it into his wife’s vagina, who be-
came pregnant. The first published reference
to donor insemination was made by Paolo
Mantegazza in 1887 (Alfredsson et al., 1983),
a pathology professor who established the first
semen bank for veterinary and possibly, for
human use (Traina, 1980). Heape (1897) and
others in several countries reported successful
AI based on studies with rabbits, dogs, and
horses. AI was first established as a practical
procedure in 1899 by Ivanov in Russia. By
1907, Ivanov had already studied AI in do-
mestic farm animals, dogs, foxes, rabbits and
poultry. He was the first to develope semen
extenders and trained technicians to select su-
perior stallions and multiply their progeny
through AI (reviewed by Foote, 2002). Some
of his investigations, especially in horses,
were included in a paper published in 1922 in
the Journal of Agricultural Science (Ivanov,
1922). Later, Milovanov (1964) established
projects for sheep and cattle breeding. The in-
vestigations carried out in Russia on AI en-
couraged other countries to take this technolo-
gy to the rest of Europe. AI in Asia started in
Japan with Nishikawa in 1912 (Nishikawa
1962, 1964) and in the United States in the
1930s. At that moment the procedures devel-
oped in assisted reproduction in animals be-
came a worldwide practice (Salisbury et al.,
1978) (Figure 1).
Porcine AI also started in Russia with
Ivanov in the early 1900s (Ivanov 1907,
1922). The technique quickly spread to the
United States (McKenzie, 1931), Japan (Niwa,
1958) and Western Europe (Polge, 1956). In
the mid 20th century extensive AI technology
in swine lead to standardization of the proto-
cols used by farmers and technicians to carry
out the process. The boar were trained on
mounting dummies (Polge, 1956) impregnated
with sow odor enabling the semen to be ex-
tracted from the boar without requiring the
presence of a sow. In addition, artificial vagi-
nas helped improve the work of collecting se-
men and safeguard sample hygiene and quali-
ty (reviewed by Althouse and Lu, 2005). The
first artificial vaginas were very similar to
those currently used, providing a means of ap-
plying pressure to the glans (McKenzie, 1931;
Polge, 1956). The gloved hand technique was
developed later by Hancock and Hovel (1959)
(see Figure 1).
Another advance in porcine AI was the
use and development of semen extenders and
frozen semen. The first diluters as a method to
store semen were developed in Russia
(Ivanov, 1922; Milovanov, 1938) and in the
United States (Philips and Lardy, 1940) with
the main objective being to use less sperm cell
per insemination. They were based on glucose
solutions with sodium potassium tartrate or
sodium sulfate and peptone, keeping the con-
centration of electrolytes low and enabling
storage of semen during long enough for
shipment and later use in the field. At that
moment, the recommended storage tempera-
ture was 7º to 12º C; however, Ito et al. (1948)
recommended storage at 15º to 20º C, as is
used at present. The most widely used semen
dilution medium is Beltsville Thawing Solu-
tion (BTS), which was developed by the la-
boratories of The United States Department of
Agriculture by Pursel and Johnson in 1975.
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70
BTS increases the storage period of fresh se-
men up to 48 h while maintaining the same
level of fertility of sperm. This and rapid
transportation of the dose represent a very im-
portant commercial advantage for producers
of pig semen.
Between the 1970’s and 90’s the re-
sults of AI in pig production improved very
strongly. The greater knowledge of the repro-
ductive physiology of both sow and boar,
knowledge of the estrous cycle of the sow and
the optimal time of insemination, the training
of technicians responsible for inseminating,
and the correct use of diluted semen have led
to similar results to those obtained with natu-
ral reproduction. The swine industry has en-
deavored in recent years to find ways to opti-
mize AI, making more efficient use of semen
and using males of high genetic value. The
development of new insemination methods
has the goal of reducing the number of sper-
matozoa needed (see section AI methodolo-
gies), and some of these techniques are cur-
rently being applied under farm conditions
(Hernández-Caravaca et al., 2012).
Figure 1. Relevant historical events in the development of AI technology.
AN OVERVIEW OF THE PORCINE
FEMALE REPRODUCTIVE
APPARATUS
The swine female reproductive appa-
ratus (Figure 2) is a long organ compared with
other species, including human, cows or even
mares. From cranial to caudal, it is composed
of a pair of ovaries to generate oocytes and
hormones such as progesterone and estrogen.
Each ovary is surrounded by a thin membrane
called the infundibulum, which acts as a fun-
nel to collect oocytes and redirect them to the
oviduct. The oviduct is about 15-25 cm long
and acts as the fertilization site, being divided
into four functional segments: the infundibu-
lum (as we already mentioned), the ampulla,
the isthmus and utero-tubal junction (Hunter et
al., 1998) (Figure 2). The utero-tubal junction
is the connection between the oviduct and
uterine horns. Uterine horns have a length of
50-100 cm in non-pregnant sow. They act as a
duct for sperm to reach the oviduct and are the
site of fetal development. The uterine body,
which is small compared with some other spe-
cies, is located at the junction of the two uter-
ine horns. The cervix is a muscular junction
between the vagina and uterus and this has
two regions: a uterine region characterized by
the presence of glandular acini, and a vaginal
region with a large vascular network. Both re-
gions showed a mixed secretory activity by
epithelial cells, which produce sulfated mu-
cins (mucous secretion) and intermingled with
abundant glycogen-rich cells (serous secre-
tion) (Rodríguez-Antolín et al., 2012). This is
the site of semen deposition during natural
mating and traditional AI (Figure 2). It is di-
lated during heat (estrus) but constricted dur-
ing the remainder of the estrous cycle and dur-
ing pregnancy. The vagina extends from the
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71
cervix to the vulva and serves as a passageway
for urine and the piglets at birth.
SPERM TRANSPORT THROUGH THE
FEMALE REPRODUCTIVE TRACT
The process of sperm transport from
the cervix (site of insemination) to the ampulla
(site of fertilization) is complex and involves
dynamic interactions between spermatozoa
and the female genital tract. This interaction
ensures the arrival of fertilization-competent
spermatozoa within the functional lifespan of
the ovulated egg. Among the factors regulat-
ing the transport of sperm in the female tract
are included mating behavior, the seminal
plasma, the spermatozoa, the female reproduc-
tive tract (musculature, secretions, epithelial
cell surfaces), the products of ovulation (oo-
cyte, oocyte investing layers, follicular fluid),
and immunocompetent elements of the female
reproductive tract (revised by Drobnis and
Overstreet, 1992).
Semen deposition from cervix to
utero-tubal junction: Billions of spermatozoa
are deposited into the cervix (during mating or
common AI), but only thousands are found in
the oviduct. Approximately 1-3x105 sperma-
tozoa reach the utero-tubal junction and about
1-3x103 reach the sperm reservoir in the cau-
dal part of the isthmus (Mburu et al., 1996).
Sperm transport to the site of fertiliza-
tion is thought to be a combination of both
passive and active transport. Passive transport
is more important in the initial phase of sperm
transport, from the site of deposition to the
proximal uterus and the utero-tubal junction
(Scott, 2000). The passive part of sperm
transport is probably due to the flow of fluid
caused by gravity and by contractile move-
ment of the uterine horns, and requires a min-
imal volume of inseminate during AI (Baker
et al., 1968). Although contractions of the my-
myometrium are vigorous during oestrus, and
should assist transport and redistribution of
the semen between the two uterine horns, an
initial distribution of semen in the uterus may
be achieved as a result of the force of ejacula-
tion and the volume of fluid involved (Hunter,
1982). Thus, the high volume of semen depos-
ited during natural mating (or in some cases
during AI) may favor displacement of a por-
tion of the ejaculate to the region of the utero-
tubal junction, which is bathed in a sperm
suspension by the completion of mating
(Hunter, 1982). Besides, the biochemical con-
stituents of seminal plasma, such as prosta-
glandins, can stimulate smooth muscle activity
of the female reproductive tract and thereby
assist the distribution of semen or spermato-
zoa within the tract (Robertson, 2007). The
mechanical stimulus of mating may also en-
hance visceral contractions and sperm distri-
bution, although the mere presence of a boar
during insemination is enough to stimulate
uterine activity through the release of oxytocin
(Langendijk et al., 2005).
After mating, sperm are transported to
the oviduct of pigs faster around the time of
ovulation than after mating earlier in estrus
(Hunter, 1991). The spermatozoa should ar-
rive in the oviducts within minutes of mating
or AI. This rate of transport is much faster
than sperm swimming speeds (active
transport); consequently, it is attributed to
muscular contractility of the female tract and
attendant changes in intraluminal pressures
(see Hunter, 2012). However, these rapidly
transported spermatozoa, will not contribute to
the fertilizing population in the oviduct (Over-
street and Cooper, 1978). Later, motile sperm
will gradually pass through the utero-tubal
junction to establish a tubal population capa-
ble of fertilizing.
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72
Figure 2. Anatomy of the sow´s uterus and sperm deposition sites during AI. The insemination sites are
shown in several real images: 1) Cervical AI (CAI) (1A and 1B: external and internal view of the cervix,
respectively); 2) Post-cervical AI (Post-CAI) (external view uterine body); 3) Deep intrauterine insemina-
tion (DUI) (external view of the uterine horn); 4) Intraoviductal AI (ITAI) (view of the laparoscopic ap-
proach of the oviduct).
Active sperm transport, resulting from
the intrinsic movement of sperm cells is prob-
ably important because it acts to keep sperm
in suspension in fluids of the female tract,
thereby reducing the scope for adhesion to the
endometrium and for migration of sperm cells
from the proximal uterus into the utero-tubal
junction and the oviduct (Langendijk et al.,
2005). In a previous report (Gaddum-Rosse,
1981) it was shown that neither immotile
spermatozoa nor a dye solution were observed
to pass through the utero-tubal junction, and it
was concluded that sperm motility is im-
portant, and probably essential for sperm entry
into the oviducts. There is some evidence that
sperm pass through the utero-tubal junction
into the isthmus via self-propulsion (see
Hunter, 2012).
Sperm reaching the storage reservoir:
The oviduct plays a significant role in the re-
productive process of mammals providing a
suitable environment. This site has a selective
binding capacity, choosing the most compe-
tent sperm population for fertilization based
on certain characteristics related to morpholo-
gy, motility, membrane integrity, or cytosolic
calcium levels and training status of tyrosine
phosphorylation of proteins (revised by Holt
and Fazelli, 2010). In the caudal part of the
Soriano-Úbeda et al., 2013
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1, 1:67-97
73
isthmus, spermatozoa bind to epithelial cells
and can be stored with no reduction in their
fertilizing ability until just before ovulation.
For this reason this part of oviduct is named
sperm reservoir (SR) (Suarez et al., 1991).
Factors involved in the formation of
sperm reservoir: The binding of sperm to ovi-
ductal epithelial cells in order to create an SR
involves carbohydrate interactions present in
the oviductal epithelial cells and lectin-like
proteins on the sperm head (Suarez, 2002).
This ligand-receptor interaction is species-
specific. For example, in pigs the molecules
involved in this process seem to be galactosyl
and mannosyl residues (Ekhlasi-Hundrieser et
al., 2005), while in hamsters, sperm binding to
oviductal epithelium is mediated by sialic acid
(DeMott et al., 1995) and by galactose in
horses (Dobrinski et al., 1996). On the sperm
side, spermadhesins AQN1 and AWN which
bind to the sequences Galb1,3GalNAc and
Galb1,4GlcNAc (Dostálová et al., 1995), have
been shown to contain carbohydrate-binding
affinities, enabling them to interact with the
epithelial cells. Whatever the case the binding
is a reversible process involving different sug-
ars in all species studied (Dobrinski, 1996;
Suarez, 1998). The relative contribution of
other factors such as mucus, the chemical
properties of oviductal fluid or temperature
gradients may contribute in varying degrees to
the formation of the reservoir.
Another important aspect to take into
account is the oviductal fluid (OF). OF has an
ionic concentration, pH, osmolarity or mac-
romolecular content that vary according to the
time of the estrous cycle and oviductal region.
In the middle of the cycle the difference be-
tween the pH of the ampulla and isthmus
ranges between 0.3 and 0.7 units, increasing a
further 0.4 units at the time of ovulation
(Nichol, 1997). These variations may be of
great significance, since an alkaline pH may
influence sperm motility and training in the
proximity of the female gamete. OF also var-
ies as regards the number of proteins (Killian,
2004) and content of sulfated (hyaluronic ac-
id) and non-sulfated glycosaminoglycans
(GAG) (Tienthai, 2000).
Sperm release from the oviductal
storage reservoir: The mechanisms that in-
duce sperm release from the porcine reservoir
are still poorly known but it has been showed
that the pattern of sperm release from the SR
and their progression along the isthmus during
the period around ovulation is sequential and
probably continuous, rather than occurring in
a bulk (Mburu et al., 1996). Spermatozoa are
gradually released from epithelial binding and,
undergoing progressive hyperactivation, pro-
ceed along the isthmus to the site of fertiliza-
tion.
A loss of binding sites on the oviductal
epithelium and/or changes in sperm (capacita-
tion and hyperactivation) could be responsible
for the release of sperm from the reservoir.
This sperm release is due not only to a re-
versible loss of epithelial binding proteins in
sperm plasma membrane (AQN1) (Töpfer-
Petersen et al., 2008) but also to the modifica-
tion of glycan residues in the epithelium by
oviductal glycosidases, such changes in the
epithelium being the consequence of the
switch from follicular oestradiol to progester-
one secretion around ovulation (Hunter,
2012). The non-sulfated glycosaminoglycan
hyaluronan, a major component of the porcine
cumulus extracellular matrix, which increases
around ovulation, has also been suggested to
participate in sperm capacitation and release
from the SR (Brüssow et al., 2008). Gradients
in temperature could be another factor pro-
moting release. During and after ovulation, an
increase of temperature in the storage region
would facilitate activation and the release of
maturing spermatozoa (Hunter, 2009).
Besides, there is evidence that sperm
changes associated with capacitation are re-
sponsible for releasing sperm. During capaci-
tation, there are some modifications in the
plasma membrane, including a combination of
shedding extrinsic proteins. The modification
or loss of these proteins could be involved in
Soriano-Úbeda et al., 2013
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74
sperm release from the oviductal ephitelium.
Remodeling of the sperm surface and of the
molecular architecture within the sperm plas-
ma membrane is viewed as one feature of the
response to a peri-ovulatory influx of Ca2+
ions into bound spermatozoa (see Flesch and
Gadella, 2000; Gadella and Harrison, 2000;
Petrunkina et al., 2001) and this influx of Ca2+
initiates the sperm hyperactivation. In mouse,
change in sperm beating increases flagellar
bend amplitudes, usually on one side of the
flagellum, which causes the flagellum to beat
asymmetrically (Suarez and Ho, 2003). The
power of the increased bend amplitude can
provide the force necessary to overcome the
attraction between sperm and epithelium. It
has been showed that only hyperactivated
sperm become detached from the epithelium
(Suarez et al., 1992; DeMott and Suarez,
1992).
The durability of the sperm in the res-
ervoir depends on the time of the estrous cycle
and varies between 36 and 48 h (Hunter,
1984). Disorder in sperm transport might re-
sult in a lack of spermatozoa at the fertiliza-
tion site or in large numbers of spermatozoa,
which might give rise to a polyspermic situa-
tion (Hunter and L’Eglise, 1971).
Sperm looking for the oocyte: Once
the sperm are released from the reservoir they
are in search of their objective, the oocyte.
Apparently, sperm are equipped with a mech-
anism for turning towards the oocyte in re-
sponse to thermotactic and chemotactic fac-
tors. Because hyperactivation occurs in the
caudal isthmus, which lies a considerable dis-
tance from the site of fertilization, sperm may
already be hyperactivated when they come
under the influence of taxis signals. A temper-
ature difference of up to 2° C between the
cooler tubal isthmus and the warmer tubal
ampulla has been detected in rabbits and there
are indications that capacitated rabbit sperm
tend to swim towards warmer temperatures
(Bahat et al., 2003). Once in the tubal am-
pulla, and close to the oocyte, chemotactic
mechanisms may guide sperm closer to the
oocyte. Among substances that have been
identified as potential chemoattractants is pro-
gesterone, which is released during ovulation
(present in follicular fluid) and is produced by
the cumulus cells that surround the oocytes
(Chang and Suarez, 2010). It has been postu-
lated that [Ca2+]i increases during sperm
chemotaxis (inducing turning swimming with
asymmetric flagellar bending) (for review, see
Yoshida and Yoshida, 2011). Other compo-
nents in OF have been identified as chemoat-
tractants, such as natriuretic peptide precursor,
which modifies sperm pattern motility and en-
hances [Ca2+]i levels, whose receptor has been
recently demonstrated in mouse spermatozoa
(Bian et al., 2012). Temperature also seems to
play a role in the levels of [Ca2+]i. Tempera-
ture stimulation activates the release of the in-
ternal sperm Ca2+ store, affecting flagellar
bending (Bahat and Eisenbach, 2010).
After fertilization, any sperm remain-
ing in the female reproductive tract may be
phagocytosed by isthmic epithelial cells or
may be eliminated into the peritoneal cavity,
where they are phagocytosed (see Suarez and
Pacey, 2006).
In summary, after AI, sperm ascend
the female genital tract and with the help of
the contractions of the uterus (passive
transport) and sperm motility itself (active
transport) arrive at the site of fertilization. Of
the total number of sperm that are deposited in
the cervix, only a small proportion is able to
reach the oviduct, bind to epithelial cells and
form the SR. In this place, sperm remain until
the time of ovulation, when they are released
sequentially by different factors, which in-
volve a number of changes at the plasma
membrane of oviductal epithelium, in the in-
traluminal fluid and sperm activity.
SPERM LOSSES IN THE UTERUS
DURING AND AFTER AI
As mentioned, only a few sperm of
those deposited reach the oviduct. Most of the
sperm are lost during insemination and on
their way through the uterus. Two of the main
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75
mechanisms known to be involved in sperm
losses are the influx of leukocytes into the lu-
men of the uterus and backflow.
The uterus acts as an immunological
organ, changing according to the oestrus cy-
clic stage. These changes affect the leukocyte
populations within the endometrium (Taylor et
al., 2009). When the female is in the oestrus
stage a massive migration of leukocytes
(mainly poly-morphonuclear neutrophils-
PMNs) into the sub-epihelial stroma takes
place (reviewed by Taylor et al., 2009). Con-
tact of the semen constituents with the uterus
and cervical tissues induces a series of immu-
nological reactions and mechanisms (Schu-
berth et al., 2008). After natural mating or AI
the PMN influx into the uterine lumen and ac-
tivated PMNs bind to spermatozoa and phago-
cytose them. Given that in some aspects se-
men is a foreign material for the female organ-
ism, it seems logical to interpret many of the
immune responses as actions to eliminate such
material (Schuberth et al., 2008). Inflamma-
tion seems to be a normal process to remove
spermatozoa and bacteria, producing an ideal
environment for embryo implantation
(Troedsson, 1997; Rozeboom et al., 1998).
The influx of leucocytes into the lu-
men is enhanced within a few hours after AI,
and PMN are cleared from the uterine lumen
within 24 to 36 h following AI (Rozeboom et
al., 1999). As a consequence of the influx,
phagocytosis by PMNs substantially decreases
the number of sperm after insemination, alt-
hough the mechanism/s and the stimulus in-
volved are still unknown. Several factors, in-
cluding sperm, seminal plasma (SP) or semi-
nal extender, may be involved in the leukocyte
influx.
Rozeboom et al. (1999) demonstrated
that spermatozoa in the absence of seminal
plasma induce a great influx of PMN into the
uterus. These results agree with other reports
showing spermatozoa to be chemotactic medi-
ators of PMN migration via complement acti-
vation (Clark and Klebanoff, 1976; Troedsson
et al., 1995). In contrast, SP has been shown to
be an essential protector of spermatozoa in an
inflamed uterine environment (Katila, 2012),
reducing chemotactic and phagocytotic activi-
ty of PMN (Rozeboom et al., 1999) and sup-
porting in vitro data (Taylor et al., 2009; Li et
al., 2012). Although other authors (Rodríguez-
Martínez et al., 2010) reported that the major
SP glycoproteins (spermadhesins) induce mi-
gration of PMN into the uterine cavity of the
sow, initiating the endometrial-related cascade
of transient and long-lasting immunological
events in oestrous sows. Therefore, semen ex-
tenders may substitute the role of SP as a ve-
hicle and provider of nutrition (Katila, 2012).
But artificial extender components cause a rise
in leukocyte numbers in vivo, probably due to
irritation of the uterine epithelium (Taylor et
al., 2009).
Other factors that can influence PMN
influx into the lumen are the ovulatory status,
dose volume, number of sperm or extender
composition. Taylor et al. (2009) observed
differences in PMN migration into the uterus
between pre-ovulatory and post-ovulatory in-
seminations. Furthermore, a reduction in the
inseminate volume and the addition of caf-
feine and CaCl2 to the inseminate dose in-
creased the number of non-phagocytosed
spermatozoa in the uterus of sows 4 h after in-
semination (Matthijs et al., 2003). In the same
way a reduction in the number of inseminated
sperm decreases the relative number of non-
phagocytosed spermatozoa (Matthijs et al.,
2003).
In species such horse, pig and cattle
the onset of PMN chemotaxis by sperm is rap-
id and the duration of PMN infiltration rela-
tively short. It has been hypothesized that
PMN takes part in sperm cell selection, re-
moving superfluous, non-motile or damaged
spermatozoa (Tomlinson et al., 1992). Wheth-
er sperm cell phagocytosis is a selective or
random process is still questionable (Schu-
berth et al., 2008). As already mentioned, this
ensures effective removal of sperm and bacte-
ria and the subsequent return of the endome-
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76
trium to a normal state, ready to receive the
embryo (reviewed by Katila, 2012).
As mentioned, the sperm in the genital
tract are reduced to a low percentage of the in-
seminated number of spermatozoa within only
4 h of insemination (Matthijs et al., 2003).
One of the main factors involved in spermato-
zoa loss, rather than PMN influx, is the back-
flow of semen. During natural mating, approx-
imately one-third of the spermatozoa in the
ejaculate is lost through backflow within 2 h
after mating (Viring and Einarsson, 1981). An
increase in uterine contractility could be one
of the main factors that causes the backflow.
SP has been shown to stimulate uterine motili-
ty in vitro (Einarsson and Viring, 1973). The
most likely reason for this is the estrogen con-
tent of SP (Langendijk et al., 2005). After in-
semination, the estrogens in the ejaculate
cause an immediate release of prostaglandin
by the endometrium (Claus, 1990). Intrauter-
ine infusion of estrogens and prostaglandin
has been shown to increase uterine motility in
sows (reviewed by Langendijk et al., 2005)
and, as a consequence, the stimulation of con-
tractions can also increase the reflux of semen
(Langendijk et al., 2002a). Willenburg et al.
(2003) also observed an increase in the back-
flow amount during AI when prostaglandin
was added to the insemination dose. In anoth-
er study (Langendijk et al., 2002b) the in-
crease of uterine contractions was attained ar-
tificially by the intrauterine infusion of clo-
prostenol raising the backflow during the in-
semination and consequently reduced the
number of sperm cells in the oviducts
(Langendijk et al., 2002a). Beside the in-
creased number of uterine contractions, the
magnitude of contractility after stimulation
and the timing of stimulation related to the
time of insemination could affect semen back-
flow (Langendijk et al., 2005).
During traditional cervical AI sperm
loss in the backflow has been reported to be
25-45% (Steverink et al., 1998; Matthijs et al.,
2003), reaching 70% of the dose volume (Ste-
verink et al., 1998). In a previous report, our
group analyzed backflow when the semen was
deposited in cervical and post-cervical (uterine
body) site. The backflow, in terms of volume
(%) and sperm (%), was reduced when the
dose was infused in the uterine body rather
than the cervix (volume: 54% vs. 38% and
sperm: 25% vs. 15%) (Hernández-Caravaca et
al., 2012). Zerobin and Spörri (1972) observed
that contractions in the caudal part of the uter-
us (cervical deposition) obstructed the infu-
sion of semen. An increased frequency of con-
tractions probably delays the influx of semen
into the caudal part of the cervix and even in-
creases the risk of backflow (Langendijk et al.,
2005). No differences were found in the back-
flow when different volumes and numbers of
sperm were deposited in post-cervical position
(Hernández-Caravaca et al., 2012), in accord-
ing with other studies where the percentage of
spermatozoa in the backflow up to 60 min did
not vary as a function of the number of intrau-
terine infused spermatozoa (Mezalira et al.,
2005). However, other factors such as ovula-
tion time or sow age may be important in
backflow quantity (Hernández-Caravaca et al.,
2012). Table 1 summarizes the backflow data
collected from different reports.
Controlling the phagocytosis activity
of PMNs towards the sperm and backflow fol-
lowing insemination could improve AI effi-
ciency in this species. Moreover, new bio-
technologies such as the use of FT semen, new
methodologies for insemination or sorted se-
men, will involve the use of a low number of
sperm, so knowledge of sperm losses could
improve their effectiveness.
SPERM SELECTION IN THE UTERUS
AFTER INSEMINATION
The fact that only several thousand of
spermatozoa reach the oviduct after the depo-
sition of billions during insemination (Matthijs
et al., 2003) suggests that, besides suffering
backflow losses and phagocytosis by PMN,
spermatozoa may be subjected to a rigid selec-
tion or unspecific clearance even before enter-
ing the oviduct (Taylor et al., 2008). There are
different mechanisms along the female genital
Soriano-Úbeda et al., 2013
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77
tract that allow the progressive selection of the
most suitable spermatozoa for fertilizing, set-
ting up different sperm subpopulations. These
subpopulations are partially or completely de-
ficient in some of the aspects necessary to par-
ticipate in the different steps of fertilization
(Satake, 2006). Also, it has been demonstrated
that each male produces his particular sperm
subpopulations capable of reaching the ovi-
duct (Holt, 2009).
Under normal circumstances a low
number of spermatozoa are sufficient for ferti-
lization, and these establish themselves in the
oviduct during the first hour after insemina-
tion (Hunter, 1981). When the sperm quality
in the backflow is analyzed (Hernández-
Caravaca et al., 2012), the results show a gen-
eral reduction in sperm quality parameters in
relation with the original sperm dose. These
results suggest that spermatozoa are already
subject to a pre-selective process within the
uterus before further selection at the utero-
tubal junction and in the oviductal isthmus.
These data agree with those of a previous re-
port (Taylor et al., 2008), in which the sperm
population was studied in ex vivo conditions
by the incubation of spermatozoa in different
fractions of the uterus. While the binding of
viable sperm to the oviduct is thought to act as
a SR, the retention of sperm cells in the uterus
could serve to protect the viable spermatozoa
from being removed with the backflow or to
help sperm maturation (Taylor et al., 2008), so
these findings could be interpreted as a pre-
selection process. Recently, Soriano-Úbeda et
al. (2013) demonstrated that uterus sperm se-
lection is not specific during the first moments
after insemination (0-15 min), but it is later
(16-60 min) that the uterus selects sperm of
better motility, discarding the less competitive
in the backflow.
These findings agree with reports in
other species such as the ruminants where, the
cervical crypts and grooves, aided by mucus,
filter defective and immotile sperm, protect
sperm from phagocytosis, act as safe storage
areas and provide privileged paths for the
transport of viable sperm (Mullins and Saacke,
1989). There are only a few reports about
sperm selection in the female track, so further
studies should be performed to clarify how the
sperm are selected along the uterus on their
way to the oviduct.
AI METHODOLOGIES
Estrus is the period around ovulation
in which sows show a standing response, al-
lowing the boars to mate with the females.
The duration of estrus varies among sows
from 24 h up to 96 h. The moment of ovula-
tion after onset of estrus also is highly variable
(from 10 h to 85 h). A reliable prediction of
ovulation time would be worthwhile, since
fertilization results are highly dependent on
the moment of insemination relative to the
moment of ovulation. When the interval be-
tween insemination and ovulation is from 0 to
24 h, fertilization is optimal (Soede et al.,
1997). A prerequisite for optimal sow fertility
is insemination with fresh extended semen
during the 24 h period before ovulation. How-
ever, the large individual variation (both in
gilts and sows) of the onset of estrus to ovula-
tion interval limits the possibility to insemi-
nate, in most of the cases, close to the optimal
time (Steverink et al., 1999) Gilts show a
shorter duration of estrus than sows, therefore
it is recommended that the time of gilt insemi-
nation, based on the onset of estrus, should
differ from that for sows. Kaneko and Koketsu
(2012) showed that it was a good standard
procedure to perform first insemination “im-
mediately” after estrus detection and to per-
form second insemination “6 to 12 h” after
first estrus detection. As mentioned, the opti-
mal time of insemination in sows is 0 to 24 h
before ovulation however, factors as duration
of estrus, duration of first estrus after weaning
or weaning-to-estrus interval must be evaluat-
ed to decide the number of inseminations per
estrus and at what point they should be done
(Soede et al., 1995).
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78
Table 1. Backflow data collected during and after insemination, reported by different authors.
References Sperm
deposition
Sperm
dose
Dose
volume
(ml)
Backflow
collection
time (h)
Backflow
volume
(%)
Sperm
(%) in
backflow
Sperm
treatment
Araujo et
al., 2009
Cervical 3.0 x 10
9
100 2 85.8 26.0
Intrauterine 1.0 x 109 100 2 83.2 16.4
Intrauterine 1.0 x 109 50 2 83.0 1.1
Intrauterine 0.5 x 10
9
100 2 87.8 16.1
Intrauterine 0.5 x 10
9
50 2 90.6 11.6
Hernández-
Caravaca
et al., 2012
Cervical 3.0 x 10
9
80 1 54.28±3.85 25.15±3.02
Intrauterine 1.5 x 10
9
40 1 39.39±4.14 15.88±2.24
Intrauterine 1.0 x 10
9
26 1 37.73±3.74 15.21±2.43
Matthijs et
al., 2003
Cervical 2.4 x 10
9
80 4 -- 42.5±2.8
Cervical 2.4 x 10
9
20 4 -- 31.7±1.0
Cervical 0.24 x 10
9
80 4 -- 47.5±8.7
Mezalira et
al., 2005
Intrauterine 1.0 x 109 100 1 66.4±30.8 14.6±13.7
Intrauterine 0.5 x 109 100 1 63.9±39.8 12.6±12.3
Intrauterine 0.25 x 10
9
100 1 67.8±35.0 17.1±15.7
Steverink et
al., 1998 Cervical
6.0 x 10
9
3.0 x 109
1.0 x 109
80
2.5
70±3.4
25±1.3
Willenburg
et al., 2003
Cervical 0.5 x 10
9
80 8 89.75 54
Cervical 0.5 x 10
9
80 8 94.37 38 Estrogens
Cervical 0.5 x 10
9
80 8 75.87 42 Oxytocin
Cervical 0.5 x 10
9
80 8 87.62 34 PGF2α
The main goal during mating or AI is
that an adequate population of spermatozoa
reach the site of fertilization during the peri-
ovulatory period. In natural service, an
enormous volume (~200-500 ml) and num-
ber of sperm (~20-70 billions) are deposited
in the genital tract. Among other benefits
(sanitary control, management, use of genet-
ic superior males or control of semen quali-
ty) AI was introduced in pig production to
optimize use of the male ejaculate. During
natural mating only one male can serve one
female. But with the use of the AI, approximate-
ly 20-25 females can be inseminated with one
ejaculate if the sperm is deposited in the cervix.
In the past two decades new strategies
have been developed with the idea of depositing
the semen close to the site of fertilization using
a lower volume and number of cells than usual.
These methods avoid the transit of spermatozoa
through most of the female tract, ensuring that
an optimal functional sperm population reaches
the oviduct at the time of ovulation. So, what
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79
the pig AI industry aims to do is optimize
boar ejaculation by decreasing the number
of spermatozoa inseminated per dose, while
maintaining the same efficiency in terms of
pregnancy rate and litter size as afforded by
traditional (cervical) insemination.
Cervical insemination: Cervical AI
(CAI) is the most widely used reproductive
methodology in the porcine industry around
the world. The technique is easy and simple
to apply in field conditions, and basically
consists of depositing the semen in the cer-
vix (Figure 2) using a catheter. The concept
behind it was logical and straightforward: to
simulate in vivo conditions during mating.
For this purpose, a catheter with the approx-
imate length of the boar penis and finishing
in a corkscrew shape (as the boar penis) was
designed (see figure 3A-i). Actually, there
are many different types of commercial cer-
vical catheters available with differently
shaped tips. However, little research has
been conducted to compare them in the
same study and so the use of one or another
depends on the economics and personal
preference on swine farms. Summarizing,
the swine catheters can be grouped in to
three types depending on the tipp: spiral
(Figure 3A-i), foam and multi-ring tip (Fig-
ure 3A-ii and iii). It is important to take in to
account the tip shape when insemination is
carried out.
Briefly, prior to insemination the
vulva should be cleaned and the tip of the
catheter coated with extender or non-
spermicidal lubricant. The catheter should
be inserted into the vagina at a 45º angle to
avoid its introduction into the urethra. Slide
the catheter until feels a resistance, indicat-
ing that the catheter is at the entrance to the
cervix. At this point, there are two manners of
insert the catheter, depending on the tip. In the
case of spiral type, turn it counter-clockwise un-
til it locks into the cervix. With a foam or multi-
ring tip just slide it (exerting some pressure) into
the cervix until it locks. Once insemination has
been made, the catheter is removed clockwise in
the first case (spiral tip) and pulled softly out-
ward in the others (foam and multi-ring tip).
Numerous investigations into the effi-
ciency of CAI have been developed. In 1992, a
study was conducted to examine the effects of
mating by natural service and AI (Flowers and
Alhusen, 1992). For this purpose the insemina-
tions were carried out twice every estrus for
each female. In the case of AI the concentration
dose was 7 billion sperm in 60 ml. When the AI
was applied the reproductive parameters ob-
tained were similar or even better than by natu-
ral service. Knowing the efficiency of AI, sev-
eral studies have focused on analyzing the ade-
quate number of sperm and volume for cervical
insemination. Watson and Behan (2002) com-
pared three different sperm concentrations (1, 2
and 3 x 109 in 80 ml of extender). When 2x109
sperm were inseminated the results were similar
to those obtained with 3x109 spermatozoa.
Pregnancy and farrowing rates and litter size
dropped drastically when the lower sperm con-
centration (1x109) was used. In addition to the
number of sperm, the fluid of the inseminated
dosage is an important factor to take in account
for an adequate fertilization rate. Baker et al.
(1968) inseminated gilts with a constant number
of sperm (5x109) but in different dose volume
(20, 100, and 200 ml). The authors concluded
that using 100 ml during the insemination ob-
tained a higher proportion of fertilized eggs and
sperm attached than females inseminated with
20 and 200 ml.
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80
Figure 3. Catheters used in swine AI. (A) Different tips used in cervical catheter insemination: i) spiral;
ii) foam and iii) multi-ring tip. (B) Post-cervical catheter: i) flexible cannula of approximately 72 cm in-
serted into a conventional cervical catheter; ii) post-cervical catheter placed in ex vivo uterus, and iii)
swine insemination using post-cervical catheter. (Images provided by Import-Vet S.A. Spain).
So, taking into account the results ob-
tained in CAI, females are commonly insemi-
nated using 2 to 3 billion sperm cells in a 80-
100 ml volume. The reproductive results ob-
tained by Hernández-Caravaca et al. (2012)
using these concentrations (3x109 sperm/80
ml) were 89% pregnancy rate, 82% farrowing
rate and 13.65 total born litter size, very simi-
lar to those obtained by other authors (91%
farrowing and pregnancy rates, and 12.5 total
born litter size) (Watson and Behan, 2002).
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81
Post-cervical insemination: As men-
tioned above, females are usually inseminated
2-3 times during estrus with 2-4 billions sperm
cells per dose, so that 4 to 12 billion sperm
cells are used per female in each estrus. These
conditions limit the number of doses that can
be prepared from one ejaculate. Various ef-
forts to perform AI have been made by con-
trolling the ovulation time, adding products to
the dosage, etc. But, recently these efforts
have been directed at reducing the number of
sperm inseminated per dose and placing the
sperm in different parts of the female repro-
ductive tract rather than in the cervix. One of
these techniques developed in the last decade
is named post-cervical artificial insemination
(post-CAI) (or intrauterine insemination) (Gil
et al., 2000; Watson and Behan, 2002; Roze-
boom et al., 2004; Mezalira et al., 2005; Rob-
erts and Bilkei, 2005; Hernández-Caravaca et
al., 2012), which consists of depositing the
sperm in the uterine body, after the cervix and
just before the uterine bifurcation (Figure 2
and Figure 3B-i).
Several studies have been made to de-
fine the most suitable conditions (mainly
number of sperm and dose volume) for this
technique to reach at least similar results to
CAI. First, let us look at some differences in
the procedure between CAI and post-CAI
methods (Hernández-Caravaca et al., 2012).
Post-CAI is performed with a combined cathe-
ter-cannula kit which consists of approximate-
ly a 72 cm long flexible cannula (15-20 cm
longer than the common one) inserted into a
conventional cervical catheter (Figure 3B).
Unlike the CAI method, the sperm dose
should be introduced quickly (only a few se-
conds) to spread the dosage through the uter-
ine horns, instead of several minutes used in
the cervical method. The inner catheter is re-
moved and then, with the cervical catheter still
placed in the cervix and shaken in a rotational
way, the neck of the womb is massaged for
five seconds, after which the catheter is re-
moved; this seems to stimulate ovulation.
A field study into the application of
post-CAI was carried out by our group (Her-
nández-Caravaca et al., 2012) in which more
than 5000 females were inseminated. Three
experimental groups were used for compari-
son: CAI (3x109 sperm cells/80 ml), post-CAI
1 (1.5x109 sperm cells/40 ml), and post-CAI 2
(1x109 sperm cells/26 ml). The results indicat-
ed that by using 1x109 sperm in 26 ml of ex-
tender for post-cervical insemination similar
reproductive rates (in terms of pregnancy, far-
rowing and total and live born litter size) are
obtained to when using common cervical in-
semination (3x109 sperm in 80 ml). Addition-
ally, when the females were post-cervical in-
seminated with 1.5x109 sperm, the results
were even better. The same results have been
reported by other authors (Watson and Behan,
2002). These data were confirmed when a
similar number of sperm were found in the
crypts and in the caudal isthmus region of the
oviducts of sows inseminated by post-CAI
(1x109 sperm) to those observed after conven-
tional AI (3x109 sperm) (Sumransap et al.,
2007; Tummaruk and Tienthai, 2010).
Other groups have attempted post-CAI
using 0.5x109 sperm per dose with controver-
sial results. On the one hand, with some au-
thors (Gil et al., 2004; Mezalira et al., 2005;
Araujo et al., 2009) found that using 0.5x109
sperm with the post-cervical technique pro-
vided a similar results to CAI or post-CAI us-
ing a higher number of sperm. On the other
hand, other authors (Rozeboom et al., 2004)
reported a decrease in the farrowing rate and
litter size when 0.5x109 sperm were used in
post-CAI in comparison with CAI group.
The application of post-CAI in field
conditions implies several advantages. One of
these is the use of a lower number of sperma-
tozoa per dose, which increases the number of
insemination doses produced per male. In cur-
rent commercial conditions, one boar can pro-
duce up to 2000 doses per year with 3 billion
sperm cells (Mezalira et al., 2005). By reduc-
ing the sperm number to 1000 million per
dose, using the post-CAI method, the number
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82
of doses can be increased by up to 300%. In
addition, the number of boars per farm could
also be reduced, saving on the costs associated
with buying and maintaining those (Hernán-
dez-Caravaca et al., 2012). In our recent report
(Hernández-Caravaca et al., 2012) which rep-
resented in-depth economic study comparing
the use of post-CAI and CAI on the farms, we
concluded that the use of the post-CAI method
would ensure important savings.
Another point in favor the post-CAI
technique is the time. CAI needs to be carried
out more slowly than post-CAI (2.76 ± 0.63
min vs. 1.12 ± 0.05 min, respectively) (Her-
nández-Caravaca et al., 2012) mainly because
of the lower volume used in post-CAI, where
the dose influx can be very fast (few seconds)
because the folds of the cervix are not a prob-
lem, and the sperm are released close to the
fertilization site. In addition when the CAI
method is used the catheter must remain in the
uterus an additional few minutes after insemi-
nation to minimize backflow. Moreover, post-
CAI insemination is straightforward and can
be performed by the own farm technicians.
Watson and Behan (2002) in their report con-
cluded that the application of post-CAI in
swine is simple, effective, and safe.
Post-CAI has been applied in sows as
well as in gilts (Dimitrov et al., 2007; Araujo
et al., 2009). However, and from our experi-
ence, the use of this methodology in gilts is
not as effective as in sows due to the physical
impossibility which presents, in some cases,
penetration of the post-cervical inner catheter
in this type of female. However, new com-
plementary methods can be used to enhance
the use of post-CAI in gilts; for example, the
application of Monzal® (Hidroclorhide of
vetrobutin, Boehringer Ingelheim), a medica-
ment routinely used to relax the uterine mus-
cle during farrowing. The administration of
this drug prior to post-CAI improves inner
catheter penetration through the cervix in gilts
(Hernández-Caravaca et al., 2013).
Deep intrauterine insemination: As
mentioned several times through the present
review, only a few of the total number of
sperm deposited will reach the oviduct. Ac-
cordingly, some researchers have thought
about the possibility of depositing only a few
thousand sperm in a place close to the fertili-
zation place, the oviduct, in a technique de-
nominated deep intrauterine insemination
(DUI) (Figure 2). This insemination was used
for the first time by Krueger et al. (1999). In
this case a surgical DUI was performed using
a very low number of sperm (between 1 and
500 million) deposited close to the utero-tubal
junction showing very encouraging results.
The next step in DUI was the use of non-
surgical insemination. For this purpose, Mar-
tínez et al. (2001) developed an optic fibre en-
doscope technique for non-surgical deep in-
trauterine insemination without sedation of the
animal. But the problem with this technique
was mainly the cost of the endoscope and im-
possibility of using it in field conditions. So,
the same authors designed a new catheter con-
structed on the basis of the endoscope used
previously (length 1.80 m, 4 mm outer diame-
ter, and 1.80 mm diameter inner tubing) but
less expensive than the endoscope. Briefly,
deep uterine catheterization is performed after
the insertion of a commercial AI spirette (to
produce a cervical lock). The DUI catheter is
then inserted through the spirette, moved
through the cervical canal, and propelled for-
ward along the uterine body and uterine horn
(reviewed by Vázquez et al., 2008).
Although the sperm dose can be re-
duced to 150 x 106 (20-fold reduction) with
the same pregnancy rate as in cervical insemi-
nation, the litter size is reduced. This reduc-
tion in fertility represents a potential economic
loss that must be considered in the total busi-
ness model when using DUI in field condi-
tions (Vázquez et al., 2008).
Other limitations of the technique for
its application in field conditions are: 1) the
high cost of the pipette for this procedure and
the difficulty of executy the technique still
Soriano-Úbeda et al., 2013
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83
represent impediments for its implantation on
commercial farms (Da Costa et al., 2011); 2)
the possibility of uterine injury (Bathgate et
al., 2008) due to the anatomical complexity of
the sow’s genital organs; 3) the risk of infec-
tion (Carabin et al., 1996). At present the ap-
plication of DUI is limited in field conditions
but is very useful for using semen from supe-
rior boars or in new biotechnologies involving
sex-sorted semen, FT or genetically modified
sperm (sperm mediated gene transfer, SMGT)
(García-Vázquez et al., 2011).
Recently, a new insemination device
was developed and named double uterine dep-
osition insemination (DUDI) (Mozo-Martín et
al., 2012). This combines aspects of post-CAI
and DUI, resulting in the post-cervical deposi-
tion of semen and approximately half-way
along the uterine horn. When tested in field
conditions this system provided similar fertili-
ty results to cervical AI when 750 million
sperm in 30-50 ml was used (Mozo-Martín et
al., 2012). Although the technique has provid-
ed encouraging results, further experiments
are required, comparing DUDI with post-CAI
and DUI methods.
Intraoviductal insemination: Another
technique that permits a drastic reduction in
sperm number during insemination is deposi-
tion directly into the oviduct (Figure 2) by
laparotomy. This method is called intratubal
artificial insemination (ITAI) and was first
used during the 1970s by Polge et al. (1970),
resulting in successful pregnancies.
Nowadays, this technique is undergo-
ing resurgence through the application of new
biotechnologies such as laparoscopy. The use
of laparoscopy instead of laparotomy offers
some advantages: 1) laparoscopy is considered
a less invasive technique than laparatomy for
introducing the semen into the uterus or in the
uterine tuba (Vázquez et al., 2008); 2) it caus-
es less stress and there is no problems of ad-
herences in the postoperative period (Fantinati
et al., 2005); 3) the procedure is relatively fast
(approximately 15-20 min per animal) (Fanti-
nati et al., 2005; Vázquez et al., 2008). The
laparoscopic ITAI method has permitted a re-
duction in sperm number deposited to 0.3-
1x106, while obtaining good oocyte penetra-
tion rates (Vázquez et al., 2008). However,
this technique is still far from being commer-
cially applied in swine. There are several dif-
ficulties involved in its application such as the
need for trained personnel, equipment costs,
risk of polyspermy (Hunter 1973; Vázquez et
al., 2008) and, in addition, the insemination
should be realized in both uterine horns be-
cause the low concentration and the small vol-
ume used prevent sperm migration to the col-
lateral horn (Fantinati et al., 2005).
NEW REPRODUCTIVE
TECHNOLOGIES FOR AI
Due to the large expansion of AI in the
swine industry in recent decades, it has be-
come necessary to implement new methodol-
ogies and improved protocols. We are entering
a new era where the application of current AI
techniques in conjunction with the application
of the newest scientific advances in assisted
reproduction is taking a great importance. This
should make available to breeders and veteri-
narians different technologies that, inde-
pendently or jointly, should increase farm
profits.
Liquid stored semen: In swine repro-
duction, around 99% of AI performed around
the world are carried out with extended liquid
semen (Wagner and Thibier, 2000) due to the
fact that the best results so far obtained in
swine assisted reproduction have been ob-
tained with fresh semen and liquid stored se-
men (Graham et al., 1978; Johnson et al.,
1981, 2000; Johnson, 1985). To prepare se-
men for insemination an appropriate extender
is necessary (Levis et al., 2000). The precise
extender composition and characteristics (sug-
ars, proteins, pH, ionic strength, osmotic pres-
sure, antibiotics, etc.) are very important for
successful insemination. The extender has to
preserve the spermatozoa integrity and fertiliz-
ing potential, but is still far from being under-
stood (Centurion et al., 2003). Nowadays, the
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84
most widely used semen extender is the Belts-
ville Thawing Solution (BTS), developed by
Pursel and Johnson in 1975 for use with fro-
zen semen, and later adapted for liquid storage
(Johnson et al., 1988).
Due to the high sensitivity of boar
sperm, semen extenders have to overcome det-
rimental consequences such as cold shock,
poor conditions of suspension medium, dilu-
tion effect and duration of storage. Tempera-
tures above body temperature or below 15º C
can drastically affect sperm viability and fer-
tility (Johnson et al., 2000). However, if sam-
ples are maintained above 15º C for several
hours prior to dilution, the spermatozoa turn
gradually resistant to cold shock (Pursel et al.,
1973). This cold shock seems to be related to
the lipid composition of the plasma mem-
brane, the low percentage of phosphatidylcho-
line and cholesterol and the high percentage of
phosphstidylethanolamine and sphingomye-
line in boar sperm (Watson, 1996). Mammali-
an spermatozoa are very sensitive to excessive
dilution, producing loss of cell viability, and
boar is not an exception. Watson (1995) pro-
posed that dilution effect is due to the exces-
sive dilution of the protective agents in semi-
nal plasma.
The key advantage in using semen ex-
tender is the possibility of storing semen in
buffers for up to a week at near room tempera-
ture (Gerrits et al., 2005). Semen that has been
extended in the liquid state can be used on the
same day or stored at 15-20º C for 1 to 5 days
(Johnson et al., 2000; Levis et al., 2000; Ger-
rits et al., 2005). Although this is an ad-
vantage, it is still far from satisfing the need to
conserve sperm beyond seven days, while
maintaining its fertility level, something that is
especially important from a commercial
standpoint and in genetically valuable ani-
mals.
Additional extender components: Be-
sides semen dilution, another strategy to in-
crease the yield of insemination is supplemen-
tation of the semen dose with several sub-
stances at the time of AI. These strategies are
especially important during summer, when re-
productive efficiency is reduced (MacLean,
1969; Auvigne et al., 2010). The addition of
PGF2α (Henze and Jurk, 1986; Waberski,
1997), oxytocine (Baker et al., 1968; Peña et
al., 1998a) or estrogens (Claus et al., 1989;
Willenburg et al., 2002) to boar sperm has
been used to improve AI.
Although PGF2α has no effect on
sperm metabolism, the addition of PGF2α to
seminal doses increase conception and farrow-
ing rates and litter size (Henze and Jurk, 1986;
Kos and Bilkei, 2004). Exogenous PGF2α
causes rhythmic uterine contractions that pro-
mote sperm transport to the utero-tubal junc-
tion (Bilkei et al., 1995). In 2000, Peña et al.
reported that the addition of 5 mg of PGF2α to
insemination doses of 119 sows during the
seasonal infertility period reduced to half the
percentage of return to estrus and significantly
increased the litter size per farrowing from
8.53 to 10.83 piglets born. In this sense, better
results have been obtained with direct vulvo-
mucosal injection of 5 mg of PGF2α. The far-
rowing rate and litter size significantly in-
creased (by 24% and 1.66 piglets per farrow-
ing, respectively), and the return to estrus rate
decreased by 25.4 % (Peña et al., 1998b). In
addition, Peña et al. (1998a) reported that
treatment with oxytocine during periods of
seasonal infertility improved litter size (2.24
and 1.94 piglets more when oxytocin was
added to the insemination dose and oxytocin
was injected in the vulvar lips, respectively)
and farrowing rates (18.36% higher with se-
men dose supplementation).
Another possibility is the addition of
estrogens to the seminal doses, because this is
physiologically present in high concentrations
in boar semen (Claus et al., 1990) and causes
the release of endogenous PGF2α that pro-
motes myometrial contractions (Langendijk et
al., 2002b). Willenburg et al. (2003) reported
an increased total number of fetuses and num-
ber of healthy fetuses in response to estrogen
addition to semen and a trend for a greater
Soriano-Úbeda et al., 2013
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1, 1:67-97
85
number of sperm to be retained in the vicinity
of SR.
Besides these hormones, other sub-
stances have been added to the fresh diluted
semen to improve the quality of the insemina-
tion doses and sperm function. In this sense, it
has already been reported that supplementing
semen doses with antioxidants (vitamines E
and C, superoxide dismutase, sodium py-
ruvate, epigallocatechin-3-gallate) improves
sperm function, reduces lipid peroxidation, in-
creases sperm vitality, motility and acrosome
integrity and increase the protective effect of
seminal plasma (Merkies et al., 2003; Córdo-
va-Izquierdo et al., 2007; Vallorani et al.,
2010).
However, the use of these substances
is quite controversial, since the advantageous
results obtained in fresh semen are not entirely
improving. Today, the application of these an-
tioxidants in fresh semen is reduced to exper-
imentation because it is not yet incorporated in
swine reproduction under field conditions.
The use of these substances is much more de-
veloped in cryopreserved semen, as is de-
scribed later in this review, in which they have
been reported to have more advantages as neu-
tralizing reactive oxygen species (ROS) pro-
duction during the cryopreservation process
(Zhang et al., 2012).
Encapsulated spermatozoa: Boar
sperm encapsulation is a technique that pro-
duces spermatozoa surrounded by a semi-
permeable membrane (Nebel et al., 1985) that
allows the exchange of nutrients, ions and me-
tabolites with the medium (Torre et al., 1998;
Ghidoni et al., 2008). This technique involves
an enclosing process, in which many sperma-
tozoa are surrounded by a single or a multi-
layered microcapsule that physically isolates
the sperm from their environment but allows
them to continue their metabolism when ex-
posed to different environments, as occur on
their way through the uterus.
These microcapsules have the ad-
vantage of allowing the gradual release of
sperm in the female genital tract for hours or
even days. Faustini et al. (2004) observed that
boar spermatozoa, encapsulated within prota-
mine-barium alginate microcapsules, are slow-
ly released under in vitro conditions for 72 h
of storage at 18º C, and that the released
spermatozoa maintained their fertility poten-
tial and a significantly higher motility. Other
advantages of this technique are that it is pos-
sible to reduce the dose of semen when a sin-
gle AI is performed (Vigo et al., 2009). The
microcapsule can also provide protection
against phagocytosis in the female genital
tract and can be used with sexed and/or FT
semen (Faustini et al., 2012). It has been re-
ported that encapsulation in alginate mem-
branes reduces the in vitro polyspermy rate in
a statistically significant way and minimizes
spermatozoa damage, with the limited disper-
sion of seminal plasma constituents preventing
precocious acrosomal reaction and hyperacti-
vation process and solving the problem of the
dilution effect (Faustini et al., 2012). Thus,
applying this promising new methodology
could save heat detection error in sows and in-
crease the likelihood of successful fertilization
(Chou et al., 2003), improving sow reproduc-
tive performance.
Filtered sperm: The objective of this
technique is to pass the semen samples
through chromatographic resins that increase
semen quality (Ramió-Lluch et al., 2009). It is
based on filtration by gravity and on the fact
that dead and abnormal sperm are retained in
the resin whereas the normal and live sperm
pass through it (Graham and Graham, 1990).
The resin most commonly used for the filtra-
tion of sperm is Sephadex beads. Some studies
have been performed in boar sperm, in which
the authors have observed that filtration in-
creases the percentage of viable and normal
sperm cells (Busalleu et al., 2008). Similar re-
sults have also been observed in other species
such as bull and dog (Anzar and Graham,
1993; Mogas et al., 1998). The filtered sperm
are resuspended in a semen extender and are
kept at the storage temperatures of cooled se-
men (16° C) for use in a maximum of 24 h
Soriano-Úbeda et al., 2013
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86
post-filtration (Ramió-Lluch et al., 2009).This
technique is particularly interesting in field
conditions when the good genetics of an ani-
mal that exhibits poor sperm quality needs to
be maintained. Furthermore, the use of this
technique requires minimal and affordable in-
frastructure.
Semen quality assessment: The selec-
tion of boars with a high fertility has an enor-
mous economic impact on farms. However,
few semen tests are available for use in field
conditions. Classical semen analysis (spermi-
ogram) is based in several simple analyses that
can be carried out quickly and at a low cost.
The main tests encompass the analysis of se-
men volume, ejaculated concentration, total
sperm in an ejaculate, progressive motility, %
of viable sperm, morphology, and acrosome
integrity. These factors are indicative of the
testicular function but, sometimes, these sim-
ple tests are insufficient to predict semen fer-
tility. In an attempt to improve this analysis,
new techniques have emerged that can help
with fertility prediction, such as computer-
assisted semen analysis (CASA), flow cytom-
etry and others, but most of them are expen-
sive and time-consuming, and cannot be used
under farm conditions (Gadea et al., 2004).
In the 1940s, scientists started to look
for an objective way to analyze the motility of
spermatozoa in an ejaculate because, until that
moment and still in most analyses at present,
this quality is subjectively analyzed and de-
pends, to a large degree, on the particular la-
boratory and the experience of the technician
who tests the sample. This is basically why
such techniques as CASA (Dott and Foster,
1979) provide the opportunity to carry out an
objective examination of each sample. CASA
is a computerized system connected to a digi-
tal camera, which visualizes and digitizes the
image of sperm cells and analyzes the sperm
concentration, % of motile spermatozoa and
cellular morphology and morphometric char-
acteristics, all with a high degree of repeata-
bility (Feitsma et al., 2011). The main ad-
vantage of CASA is its objectivity, but this is
only reached when it is operated properly and
by trained laboratory technicians and if the
choice of the field to be observed is random
(Feitsma et al., 2011). Significant correlations
between the basic parameters of CASA and
fertility have been described for several spe-
cies, including pigs (Holt et al., 1997; Vyt et
al., 2008). CASA, then, has the potential to
become a useful tool for optimizing semen
dose production.
Another objective semen analysis
technique is flow cytometry, whereby sperma-
tozoa are fluorescently labeled and analyzed
(thousands per second), allowing the assess-
ment of different semen quality characteristics
related to male fertility (reviewed by Gadea,
2005; Broekhuijse et al., 2012). Among these
characteristics are sperm membrane integrity,
acrosome intactness, acrosome responsive-
ness, chromatin structure and the potential of
the inner mitochondrial membrane. These data
help decide which ejaculates should be used,
how concentrated the insemination doses
should be, what the insemination dose lifespan
is and what the expected fertility is
(Broekhuijse et al., 2011). Probably, in the
near future, the development of new
knowledge in molecular and genomic technol-
ogy could help us the fertility of boar ejacu-
lates to be predicted with a greater accuracy.
APPLICATION OF AI TO ‘SPECIAL’
SPERM
Nowadays, in almost all assisted re-
productive procedures performed in swine
production worldwide fresh semen is diluted
and stored at a temperature of between 15 and
20° C (Johnson et al., 2000). This is currently
the way in which the best results are obtained
in reproductive performance on farms in terms
of farrowing rate and litter size. However, it
has been necessary to develop techniques that
allow other types of sperm to be used in an at-
tempt to improve results obtained. This is the
case of FT spermatozoa; sexed-semen and
sperm mediated gene transfer (SMGT). How-
ever, the results have not yet achieved compa-
rable levels of success to the use of fresh se-
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87
men (Johnson et al., 2000). We describe be-
low all such techniques used today and those
still under development to reveal a hopeful fu-
ture in assisted reproduction in swine under
field conditions.
Frozen-thawed spermatozoa: In an at-
tempt to improve insemination techniques, FT
semen samples emerged in the mid twentieth
century and have become commercially avail-
able since 1975 (Gerrits et al., 2005) both in
pellet form and in straws. This methodology
has a global interest for the swine industry for
the long-term preservation of genetic re-
sources, genetic improvement, for the distribu-
tion of genetic material across countries, min-
imizing boar transportation (Bailey et al.,
2008) and for the control of very important
diseases, such as porcine circovirus disease
(PCVD) and porcine reproductive and respira-
tory syndrome (PRRS) (Bailey et al., 2008).
However, currently only 1% of the insemina-
tions that take place worldwide in swine uses
FT semen (Johnson et al., 2000).
The main disadvantage of this method
is the low fertility compared to the use of
cooled semen (Johnson et al., 2000; Roca et
al., 2003). A high percentage of sperm do not
survive after thawing (40-50%) (Almlid et al.,
1988; Watson, 2000) and survivor sperm mo-
tility is much lower than that of fresh semen
(Watson, 2000). The decrease in farrowing
rate with respect to the use of cooled semen is
approximately 20-30% and litter sizes are 2 or
3 piglets smaller (Johnson et al., 1985). It is
known that boar spermatozoa are more sus-
ceptible to cold shock than other species and
an important percentage of spermatozoa die
during the freezing procedure (Martínez et al.,
2005). The effects that FT has on boar sper-
matozoa make them particularly vulnerable
during their transit through the female genital
tract, which is translated into a low sperm
count so that very few of them are able to
reach the SR. The success of fertilization is
therefore seriously compromised (Roca et al.,
2006).
FT semen is not as widely used in as-
sisted reproduction swine (Wagner and
Thibier, 2000) for several reasons: firstly, be-
cause of the low proportion of FT spermato-
zoa that reach the SR in the uterus and its
short lifespan. Secondly, because it is neces-
sary to use an insemination dose with a high
concentration of spermatozoa, making this
technique disadvantageous from an economic
point of view. And finally, because farmers
are forced to greatly increase the accuracy of
detection of sow ovulation, which add to their
routine work (Waberski et al., 1994).
However, the use of insemination
methods such as post-CAI or DUI should im-
prove the fertility results for FT semen (Roca
et al., 2006; Casas et al., 2010). Moreover, the
use of cryoprotectants such as glycerol (Polge
et al., 1949) or egg yolk (Benson et al., 1967)
is very helpful to prevent the formation of ice
crystals within the spermatozoa during freez-
ing. In addition, antioxidants such as glutathi-
one (Gadea et al., 2005), superoxide dismutase
or ascorbic acid can neutralize the excessive
production of ROS during cryopreservation
and protect spermatozoa from damaging the
cellular structure (Zhang et al., 2012). These
substances added to sperm at appropriate con-
centrations can minimize some of the negative
effects that FT has on sperm. It has been de-
scribed that the structural damage that sperm
suffer during FT is due to the loss of mem-
brane cholesterol, which causes intracellular
events that harm the spermatozoa, causing
spontaneous acrosome reactions and a capaci-
tation-like state (Bailey et al., 2008). Boar
sperm is more susceptible to this phenomenon
because it has a lower cholester-
ol:phospholipid ratio compared to the sperm
of other mammals (Bailey et al., 2008). For all
these reasons, swine semen requires special
consideration in research and in the design and
development of FT protocols.
Sexed semen: This technique opens up
a new door to progress in assisted reproduc-
tion and represents one of the greatest advanc-
es in the swine industry. It allows a specific
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88
gender (males or females) to be obtained with
desirable genetic traits and even in association
with the use of other reproductive technolo-
gies such as embryo transfer or FT sperm
(Bathgate, 2008). Sex-sorting identifies differ-
ent chromosomes of the sperm and is capable
of separating sperm according to their differ-
ing DNA content (Garner et al., 1983). Thus,
it is possible to obtain separate populations of
X- and Y-chromosome spermatozoa. For sex
separation, all the spermatozoa contained in
the ejaculate are stained with bisbencimide
(Hoechst 33342), a vital fluorescent stain that
only binds to DNA. Using a high-speed flow
cytometer, the stained spermatozoa are ex-
posed to an ultraviolet laser (UV) that is di-
rectly projected over them and a computer col-
lects the intensity of fluorescent emission of
each spermatozoon. Since the Y-chromosome
is smaller than the X-, the fluorescence inten-
sity emitted by Y-spermatozoa is lower than in
X-, and, accordingly, sperm are separated by
magnetic plates in different containers. Once
separated, the sperm are resuspended in semen
extenders.
This technique allows up to 3x104
spermatozoa per second to be identified and
separated according to their chromosomes,
and up to 15x106 sex-sorted spermatozoa per
hour, with more than 85% purity (Niemann et
al., 2003). But this precisely is the main hand-
icap of this technique, and even working 24 h
a day, only 360x106 sex-sorted spermatozoa
would be obtained (Maxwell et al., 2004),
which is economically unviable. Moreover,
there are some aspects which can damage
sperm: dilution, bisbenzimide staining and ex-
posure to UV or high pressure (Bathgate,
2008) which can seriously compromise sperm
survival or lifespan
The first piglets born from sex-sorted
fresh spermatozoa were obtained using surgi-
cal intraoviductal insemination of 3x105 sperm
by laparotomy (Johnson et al., 1991), in which
the spermatozoa were directly deposited in the
oviductal isthmus. However, this surgical
technique of insemination is impossible under
farm conditions. Later, Rath et al. (2003) re-
ported the successful pre-selection of sex after
intrauterine insemination with sexed semen.
They used DUI to deposit 50x106 fresh sex-
sorted spermatozoa in a volume of 2 ml and
finally obtained 11 piglets, which represents a
very encouraging result.
The swine industry would obtain nu-
merous economic and ethical benefits with
this technique if it were economically feasible
in field conditions. At the moment, it is only
viable in pigs in IVF or ICSI, but hopefully, in
the near future, this technique will become
available for use in swine as it currently is in
bovine or equine species.
Sperm Mediated Gene Transfer
(SMGT): Animal transgenesis is a powerful
biotechnology which has seen exponential
growth in the last two decades. This includes
several techniques to produce genetically
modified animals (Gadea and García-
Vázquez, 2010a); one of these is the sperm
mediated gene transfer or SMGT, which is
based on the ability of sperm to bind, internal-
ize and transport exogenous DNA into an oo-
cyte during fertilization (Brackett et al., 1971;
Lavitrano et al., 1989). So, the use of “trans-
genic sperm” in combination with the new AI
methodologies could improve the efficiency of
this method. Several authors have tested the
use of SMGT with DUI (García-Vázquez et
al., 2011) or laparoscopic insemination (Fanti-
nati et al., 2005).
The benefits that breeders can obtain
through this gene-transfer technique are di-
verse but include the production of healthier
pigs, animals that are less sensitive to envi-
ronmental stress or with increased resistance
to common diseases (Gadea and García-
Vázquez, 2010b). But further technical devel-
opment of SMGT is needed to achieve full
application under field conditions.
Soriano-Úbeda et al., 2013
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89
CONCLUDING REMARKS
Although porcine biotechnology is im-
proving day by day and new methods are a re-
ality on the farm, such as post-cervical insem-
ination, there is still a long way to go. Howev-
er, some techniques should be available to the
porcine industry in the near future. For exam-
ple, the use of sex-sorted sperm, whether the
number of sperm sorted is improved, or the
viability and fertility rate of FT sperm is en-
hanced. Another aspect that should see pro-
gress is fresh sperm storage; for this purpose,
investigations must focus on new extender
formulations that permit long term storage (at
least 7 days) at 15-20º C. This, in combination
with other new strategies, such as post-CAI,
would vastly increase boar effectiveness. The
development of new in vitro technologies to
predict male fertility is another theme that will
be resolved sooner or later, permitting use of
the best boars and at the most appropriate
moment. Besides these, fresh knowledge on
the physiology of reproduction is necessary
before new strategies can be widely applied in
swine-breeding practice. In this respect,
knowledge of the exact mechanism by which
sperm reach the oviduct, how sperm are se-
lected during transit in their travel or, very
ambitiously, what the special characteristics of
the spermatozoa which fertilize the oocyte are,
would provide to the reproductive industry
new opportunities enhance efficiency at farm
level and provide economic savings.
ACKNOWLEDGMENTS
The authors are very grateful to Ivan Her-
nández-Caravaca and María José Izquierdo-Rico
for their contribution to this work. They also thank
Pedro José Llamas (Import-Vet S.A. Spain) for his
technical support. Financial funding by MINECO
and FEDER (AGL2012-40180-C03-01), Funda-
ción Séneca (08752/PI/08) projects and Import-
Vet S.A. (15694) and Boehringer Ingelheim Spain,
S.A. (15317).
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