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An overview of swine artificial insemination: Retrospective, current and prospective aspects

January 2013
Journal of Experimental and Applied Animal Sciences 1(1):67-97

DOI:10.20454/jeaas.2013.709

Authors:

Cristina Soriano-Úbeda

University of Leon

Carmen Matás

University of Murcia

Francisco Alberto Garcia-Vazquez

University of Murcia

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 efficiency 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.

Relevant historical events in the development of AI technology.

…

. Backflow data collected during and after insemination, reported by different authors.

…

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 insemination (DUI) (external view of the uterine horn); 4) Intraoviductal AI (ITAI) (view of the laparoscopic approach of the oviduct).

…

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 inserted 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).

…

Figures - uploaded by Cristina Soriano-Úbeda

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Content uploaded by Cristina Soriano-Úbeda

Content may be subject to copyright.

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

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-

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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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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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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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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

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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

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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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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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1, 1:67-97

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

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1, 1:67-97

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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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

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

100 2 87.8 16.1

Intrauterine 0.5 x 10

50 2 90.6 11.6

Hernández-

Caravaca

et al., 2012

Cervical 3.0 x 10

80 1 54.28±3.85 25.15±3.02

Intrauterine 1.5 x 10

40 1 39.39±4.14 15.88±2.24

Intrauterine 1.0 x 10

26 1 37.73±3.74 15.21±2.43

Matthijs et

al., 2003

Cervical 2.4 x 10

80 4 -- 42.5±2.8

Cervical 2.4 x 10

20 4 -- 31.7±1.0

Cervical 0.24 x 10

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

100 1 67.8±35.0 17.1±15.7

Steverink et

al., 1998 Cervical

6.0 x 10

3.0 x 109

1.0 x 109

2.5

70±3.4

25±1.3

Willenburg

et al., 2003

Cervical 0.5 x 10

80 8 89.75 54

Cervical 0.5 x 10

80 8 94.37 38 Estrogens

Cervical 0.5 x 10

80 8 75.87 42 Oxytocin

Cervical 0.5 x 10

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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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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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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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).

An overview of swine artificial insemination: Retrospective, current and prospective aspects

Abstract and Figures