BN Day

C Krueger

EA Martinez

I Parrilla

JM Vazquez

LA Johnson

Peer Ola Hofmo

PF Watson

PJ Hendriksen

Springer - Publisher Connector

BioMed CentralActa Veterinaria Scandinavica
ss
files/pdf/1751-0147-48-S1-full.pdf</url> </supple-Open AcceOral presentation
Sperm sorting and low-dose insemination in the pig – an update
Peer Ola Hofmo*
Address: Norsvin Breeding Company, Hamar, Norway
* Corresponding author    
Introduction
During the last decade efforts have been made to develop
techniques for pre-selection of the sex of the offspring.
Such pre-selection may be desirable for different reasons;
production of female hybrid pigs in multiplying herds or
production male pigs for breeding reasons (e.g. AI boars
or breeding boars). These are niche areas, and application
of new techniques may therefore be done with less focus
on efficiency and economy. However, pre-selection of the
sex for use in commercial routine piglet (or slaughter pig)
production due to banning of castration requires a differ-
ent view on application of such a technique. It must be
cost-effective and easy to implement in routine semen
production. In addition, it must be possible to use sorted
semen without having to use a sophisticated insemination
technique.
This article will review the challenge concerning sperm
cell sorting and low dose insemination for use in commer-
cial pig production
Sex sorting of sperm cells
At present time, the only means of farrowing pre-selected
piglets is by sorting sperm cells according to the Beltsville
Sperm Sexing Technology (BSST) [1]. This method
includes a flow cytometer and a cell sorter, and the sorting
is based on differences in the DNA content between X and
Y chromosome bearing sperm cells. In the pig this differ-
ence is 3.6 %; the X-bearing sperm cell ("female sperm")
having the highest DNA content. This difference varies
across species, and in chinchilla the difference is as high as
7.5 % while in humans only 2.8 % [2].
The sorting process includes dilution of the semen, stain-
ing the sperm cells, identifying X and Y chromosome bear-
ing sperm cells, sorting X and Y chromosome bearing
sperm cells and recovering and storage of the sorted sperm
cells. There are several major challenges among these
operations that have to be solved before the method can
be used on a regular basis for commercial slaughter pig
production.
In the flow cytometer, laser light is used to illuminate
stained sperm cells as they pass through a laser beam, one
at a time, in a fine fluid stream. A critical part of the proc-
ess is to ensure a uniform staining intensity within the cell
population. Variation in the staining intensity will reduce
the efficiency in separating the cell populations signifi-
cantly.
Light scattered by the cells and light emitted by the fluo-
rescent dye are analysed by several detectors and proc-
essed by a computer. The X and Y bearing sperm cells are
distinguished on the basis of the DNA content. Since the
head of the sperm cell is paddle shaped, the cells must be
oriented with the flat side against the laser beam. Two
laser beams (0° and 90°) and a special needle are used so
that when the fluid stream containing the sperm cells pass
the laser beam, a high proportion of the cells will be fac-
ing the beam in the proper plane. The efficiency in distin-
guishing the two populations are depending on that a
high proportion of the sperm cells are oriented in the
proper way.
After passing the laser beam, the X and Y chromosome
bearing sperm cells are given different charge and the cells
are sorted as they pass between two continuously charged
plates. The sperm cells are sorted in three populations; X
chromosome bearing sperm cells, Y chromosome bearing
sperm cells and waste (non-sorted sperm cells). The chal-
from Prevention of Boar Taint in Pig Production: The 19th Symposium of the Nordic Committee for Veterinary Scientific Cooperation
Gardermoen, Norway. 21–22 November 2005
Published: 7 August 2006
Acta Veterinaria Scandinavica 2006, 48(Suppl 1):S11 doi:10.1186/1751-0147-48-S1-S11
<supplement> <title> <p>Prevention of Boar Taint in Pig Production: The 19th Symposium of the Nordic Committee for Veterinary Scientific Cooperation</p> </title> <note>Meeting abstracts</note> <url>http://www.biomedcentral.com/content/ment>
© 2006 Hofmo.; licensee BioMed Central Ltd.Page 1 of 3
(page number not for citation purposes)
Acta Veterinaria Scandinavica 2006, 48(Suppl 1):S11lenge in this operation is to reduce the waste, by enhanc-
ing proportion of sorted cells.
During sorting, the sperm cells are highly diluted. Once
sorting is terminated, the sperm cells must be centrifuged
(300 × g) to increase the density. The density will vary
depending on the method selected for use of the sorted
sperm cells. This process affects the viability of the sperm
cells and it has been shown that the fertilising ability of
sorted sperm cells is beginning to decrease at 5 hrs of stor-
age [3]. It is therefore necessary to deposit sorted semen as
close as possible to the ovulation time and the site of fer-
tilisation. Premature capacitation of sorted sperm cells is
one of the changes that shortens the viable lifespan for in
vivo insemination.
Concerns have been raised whether exposure to the dye
(Hoechst 33342) and the laser beam might damage the
DNA. However, there seems not to be any higher propor-
tion of abnormalities among offspring from sorted sperm
cells compared to not sorted sperm cells. Furthermore, the
mutagenic effect has been investigated without evidence
of any negative effect on offsprings from stained sorted
sperm cells [4].
State of the art from a practical point of view
To be used in commercial semen production, the speed of
the sorting as well as the purity of the sorted sperm cells
are crucial. The purity (efficiency of the sorting) will
largely depend on the speed, making these two elements
contradictory; the higher the speed, the lower the purity of
sorted sperm cells. The present technology makes it possi-
ble to sort 15 million sperm cells per hour [1]. Although
deep intrauterine insemination may reduce the number of
sperm cells required for fertilisation significantly, the
number is still too large to be considered for practical con-
ditions.
Other methods for sex sorting of sperm cells have been
discussed, and the focus has mostly been towards a possi-
ble difference in surface proteins between X and Y chro-
mosome bearing sperm cells. If so, one could produce an
antibody to attach to the X or Y chromosome bearing
sperm cells and then use magnetic beads to separate the
two populations. This method could provide a large scale
separation process easily adaptable to AI centres. Approx-
imately 1000 surface proteins have been mapped but no
difference has been detected between proteins isolated
from X versus Y chromosome bearing sperm cells [5,6].
Low dose insemination in the pig
Optimum fertilisation rate is depending on several fac-
tors; the interval between insemination and ovulation, the
quality of the sperm cells, the life span of the sperm cells
in utero and the site of deposition of the sperm cells. Tra-
ditional artificial insemination in the pig is carried out
using 2.5 – 4 billion sperm cells deposited in the posterior
part of cervix uteri. Only a very small proportion of the
sperm cells reaches the site of fertilisation (the oviduct)
due to a combination of loss through back flow and
phagocytosis of sperm cells by polymorphonuclear leuko-
cytes. It has, however, been shown that acceptable fertility
can be achieved by deposition of only 10 million sperms
at the uterotubal junction by using surgical insemination
close to ovulation [7].
Surgical insemination can not be used in commercial
farms, and during the last 5–7 years efforts have been
made to develop new devices to be able to deposit the
semen either in the uterine body or deep in the uterine
horns. The main obstacles are the cervical folds in cervix
uteri and the long uterine horns (up to 1.5 meters). How-
ever, two new procedures for deposition of the semen cra-
nial for the cervix have been developed; post cervical
insemination and deep intrauterine insemination.
Post cervical insemination
By post cervical insemination, the semen is deposited in
the uterine body, just cranial for the cervix. Several differ-
ent (commercial) catheters are available, and the aim is to
reduce the number of sperm cells. The method may be
used on a regular basis in commercial herds, and it has
been shown that it is possible to reduce the number of
sperm cells from 3 to 1 billion without compromising the
fertility [8]. However, for use in combination with sorted
semen this technique requires too many sperm cells.
Deep intrauterine insemination
Deep intrauterine insemination requires a long flexible
catheter to pass the cervix and enter the uterine horns. It
has been reported that it is possible to deposit semen close
to the uterotubal junction with a 20-fold reduction of the
number of sperm cells without affecting farrowing rate or
litter size [9]. However, by reducing the number of sperm
cells further it seems like the litter size is compromised
although pregnancy and farrowing rates are not affected
compared to traditional AI with ~3 billion sperm cells
[10.11].
Conclusion
In conclusion: It is possible to sort sperm cells in X and Y
chromosome bearing populations. The only method
proven is the Beltsville Sperm Sexing Technology which
uses a flow cytometer and a cell sorter. The sorting speed
is at present approximately 15 million cells per hour.
To be able to use sex-sorted semen in commercial pig pro-
duction the number of sperm cells per insemination must
be reduced tremendously. None of the newly developed
technologies makes it possible to reduce the number ofPage 2 of 3
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sperm cells inseminated to such an extent that sorted
semen may be used.
The major challenge in sex sorting boar spermatozoa is to
increase the speed of BSST-sorting or to develop new tech-
nologies. Furthermore, the damage caused by the sorting
must be reduced, the storage time for sorted sperm cells
must be increased and the waste (non-sorted population)
must be reduced.
References
1. Johnson LA, Rath D, Vazquez JM, Maxwell WM, Dobrinsky JR: Prese-
lection of sex of offspring in swine production: current status
of the process and its application.  Theriogenology 2005,
63:615-624.
2. Johnson LA: Sex preselection by flow cytomertric separation
of X and Y chromosome bearing sperm based on DNA dif-
ference.  A Review Reprod Fertil Dev 1995, 7:893-903.
3. Parrilla I, Vazquez JM, Cuello C, Gil MA, Caballero I, Almiñana C,
Roca J, Martinez EA: Influence of storage time on functional
capacity of flow cytometrically sex-sorted boar spermato-
zoa.  Theriogenology 2005, 64:86-98.
4. Parrilla I, Vazquez JM, Cuello C, Gil MA, Roca J, Di Berardino D, Mar-
tinez EA: Hoechst 33342 stain and u.v. laser exposure do not
induce genotoxic effects in flow-sorted boar spermatozoa.
Reproduction 2004, 128:615-621.
5. Hendriksen PJ, Welsh GR, Grootegoed JA, Van der Lende T, Johnson
LA: Comparison of detergent-solubilized membrane and sol-
uble proteins from flow cytometrically sorted X- and Y chro-
mosome bearing porcine spermatozoa by high resolution 2-
D electrophoresis.  Mol Reprod Dev 1996, 45:342-350.
6. Hendriksen PJ: Do X and Y spermatozoa differ in proteins?
Theriogenology 1999, 52:1295-1307.
7. Krueger C, Rath D, Johnson LA: Low dose insemination in syn-
chronized gilts.  Theriogenology 1999, 52:1363-1373.
8. Watson PF, Behan JR: Intrauterine insemination of sows with
reduced sperm numbers: results of a commercially based
field trial.  Theriogenology 2002, 57:1683-1693.
9. Martinez EA, Vazquez JM, Roca J, Lucas X, Gil MA, Parrilla I, Vazquez
JL, Day BN: Minimal number of spermatozoa required for nor-
mal fertility after deep intrauterine insemination in non-
sedated sows.  Reproduction 2002, 123:163-170.
10. Vazquez JM, Martinez EA, Parrilla I, Cuello C, Gil MA, Lucas X, Roca
J, Vazquez JL, Didion BA, Day BN: Deep intrauterine insemina-
tion in natural post-weaning oestrus sows.  In Proceedings of the
6th Int Conf on Pig Reprod Columbia, MO, USA; 2001. 
11. Day BN, Mathias K, Didion BA, Martinez EA, Caamano JN: Deep
intrauterine insemination in sows: first field trial in USA
commercial farm with a newly developed device [abstract].
Theriogenology 2003, 59:213.Page 3 of 3
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Sperm sorting and low-dose insemination in the pig – an update

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