Table 1. Nation Pork production. Number of sows

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1 Highlights of The 7 th International Conference on Boar Semen Preservation Bonn Germany August 14-17, Rob Knox Department of Animal Sciences University of Illinois Introduction This paper includes selected presentations from The 7 th International Conference on Boar Semen Preservation held in Bonn Germany August 14-17, 2011 and Published in the Journal: Reprod. Dom. Anim. 46: (Suppl. 2) Review on trade with boar semen Global use of AI, semen sourcing and trade was reviewed (Riesenbeck, 2011) with the extent of AI use listed for most countries with significant numbers of sows and pork production (Table 1 is an excerpt from original). Many industrialized nations with advanced infrastructure use AI at very high levels but level of use was still country dependent. Europe, North, Central and South Americas showed the majority of nations used AI on >80% of matings and in many >90%. In less developed countries overall use of AI was lower, but could be very high in modern farms located in Asia and South America and elsewhere. Many countries with lower use of AI technology will have great future potential for genetic advancement with adoption of AI technology. The number of AI centers or boar studs was available for only few countries. Some countries such as Germany had 22 AI centers with ~6500 boars and Spain had 74 AI centers with 7500 boars. For comparison, Denmark had 13 studs with 3100 boars while Vietnam had 4500 boars in over 500 studs. For international semen trade, disease concern was the most important factor with Aujusky s, CSV, and Brucellosis of greatest concern. Other limitations to trade included costs, health records and administration, short lifespan of liquid semen, and need to maintain temperature at 17 C. Several countries are currently involved in semen export to numerous countries. Table 1. Nation Pork production Number of sows AI use (%) No. boars No. Studs Semen Doses/Yr. China USA , Germany Spain Brazil Canada Vietnam France Denmark Netherlands Russia 2.0 Poland Philippines Italy Japan Mexico

2 Use of Frozen Boar sperm A review of the potential for use of FTS (Frozen-Thawed Boar Semen) by commercial companies (Knox, 2011) included opportunities and limitations of its use. Opportunities for use included improved potential to limit disease entry into genetic source herds that operate as closed systems or with limited entry or access to new animal or semen shipments. With FTS, number of new genetic entries can be increased. This can occur with minimal risk with improved testing of semen and boars prior to use and without concern for viable shelf-life before use. Further, in genetic nucleus herds, FTS would aid in maintenance of genetic families or lines by ensuring the availability of semen from proper sires to mate with selected dams. Importation and export of semen in frozen form eliminates problems with viability as a result of long transit time, shipping stresses, and handling issues compared to liquid semen. Use of FTS is accepted as a reasonable and practical application for introduction of new genes by this route. The primary limitations to FTS use involve lower pregnancy rates and litter sizes. This is more evident at the commercial farm level than the genetic source herds though. New results with FTS suggest improved fertility in swine, but not without some loss of gene transfer efficiency, reproductive performance and increased costs and labor. The ability to produce higher number of FTS semen doses with improved sperm cell fertility is required in order to minimize costs and extend boar use. Methods to facilitate this technology should include education and training and models for use. Genetic selection for freezing Selection of boars for freezing could be important for application of the technology (Safranski et al., 2011) as breed and line differences has identified good and poor freezers. Further, in chickens, selection for post-thaw fertility resulted in a longer duration of sperm fertility by 0.29 days/generation for each of 5 generations when using AI. Although the h 2 can be low (17%) and most of the differences in post-thaw quality is associated with individuals, this approach is promising. However, one concern involves whether the selection for sperm traits will result in poor correlation with highly valued production traits. A model based on over 900 ejaculates that were frozen examined correlations and h 2 of fresh with postthaw motility parameters using CASA. Fresh and frozen motility were moderately correlated (~0.3) while many motion parameters and percent live were highly correlated. Measures of h 2 for fresh semen motility (0.19) and live (0.5) and post-thaw motility (0.37) and live (0.19) were estimated. There were no significant correlations (negative or positive) of semen traits with backfat or 150 day weight gain. These results suggest that it is possible selection for freezing could improve doses produced and fertility with AI without detrimental consequences to production and that this type of approach may be well-suited for DNA marker assisted genetic selection. Processing boar sperm for freezing Sperm populations in many species can be separated based on differences in their physical properties using density gradient procedures with such materials as colloid, percoll and PVP (Morrell and Wallgren, 2011). This type of process results in different populations of sperm between boars and with resulting differences in the fertile populations of sperm. Single layer centrifugation (SLC) of sperm would be more practical and results in sperm populations with improved motility and morphology compared to unselected ejaculates. This process increases the shelf-life of sperm perhaps through removal of pathogens such as bacteria and most virus. This process could reduce the need for antibiotics in extenders. Antioxidants function to protect boar sperm from damage during the freeze-thaw process (Bathgate, 2011). Boar sperm may be more susceptible to oxidative damage than the sperm of other species due to its high concentration of unsaturated fatty acids in the sperm membrane lipids and low anti-oxidative activity of boar seminal plasma (SP). In a sperm cell, most oxygen is used for ATP energy production and 2

3 then converted to water while 1-2% is converted to one of several a reactive oxygen intermediates (ROS). At normal low levels, these ROS function in normal processes for sperm hyperactivation, capacitation, the acrosome reaction and egg binding. However, these ROS can cause problems for sperm at high concentrations as they can interact with lipids, sulfa groups, enzymes, proteins, and DNA to cause damage. More ROS are produced during the freeze and thawing process and even during storage in liquid nitrogen. There are natural antioxidants present in the activities of enzymes such as superoxide dismutase as well as in the function of other molecules such as glutathione and vitamin E. These antioxidants have been tested for their ability to protect in the form of feed supplements and by addition of these to media. Supplementation of media with many of the antioxidants was proven to be beneficial on many sperm measures but feeding approaches for boars has not yet shown the same effects on sperm measures in ejaculates. The Boar Sperm Cell During Freezing During the freezing process, boar sperm cells are modified from the original ejaculated form by diluents and processing steps that result in what is known as capacitation-like changes in the sperm cell. Capacitation is a normal process of sperm as they mature. However, cryo-capacitation or induced capacitation is an accelerated process that changes the extent and results of the process. Capacitation impacts the proteins and lipids in the sperm outer membrane (Leahy and Gadella, 2011). In-vitro capacitation of sperm can be achieved by passing sperm through a percoll centrifuge density gradient to partially strip loose factors from the sperm surface. The presence of these substances prevents capacitation while their removal allows destabilization of the membrane and sperm capacitation. The centrifugation step separates sperm into populations with different densities. More dense sperm are more matured with chromatin condensation and loss of the cytoplasmic droplet. Capacitation is a first step required for sperm to fertilize an egg. The normal capacitation changes in sperm can be mimicked in-vitro and the membrane proteins rapidly reorganized into regions that allow egg binding and inner membrane fusion and the acrosome reaction. However, during the freeze-thaw process, sub-lethal changes can occur to sperm that can include a de-coating of the membrane extracellular lipids and proteins and a re-coating with other proteins and lipids from the milk, egg yolk and albumin based extenders. Further, extensive dilution of the sperm and the dramatic temperature decline, cause a phase separation of the lipids in the membrane and a change in the membrane permeability to water and the cryo-protectant. Some of these changes can be reversed while others are lethal in the cell s ability to survive a future stress. Research has shown that addition of seminal plasma (SP) to frozenthawed sperm can alleviate some of the detrimental effects although the source of the SP is a source of high variation in response. Further, SP addition by itself shows mixed results and may be due to the presence of abundance of specific proteins. The most abundant proteins are known as the spermadhesins. These are a family of proteins (AQN-1, and 3, and AWN) which differ in their binding to heparin and also the PSP I and II proteins. Collectively, these five make up 90% of SP protein and are present in the fluids of the epididymus, seminal vesicles, as well as the female reproductive tract. The issue of these proteins is complex as these adhesins can combine and localize on the surface of the sperm in combinations and as a result single proteins may not prevent capacitation or enhance fertility. Future efforts will evaluate these proteins with respect to their known processing steps and stressors as each could change the sperm membrane make-up of each of these proteins. The effect of freezing has also been evaluated for its effect on nuclear DNA damage (Fraser et al., 2011). DNA of all species can be fragmented by the freeze-thaw process regardless of sire, extender, or packaging. Mature healthy sperm show changes in the type and amount of sperm nucleo-proteins as sperm mature in the epidiymus. These changes ensure DNA remains highly condensed. Tightly condensed DNA adds protection as there is no DNA repair mechanism in the sperm and also functions to 3

4 allows efficient DNA passage into the egg upon fertilization. Some reports reveal that the freeze-thaw process can cause breaks in DNA or even increased DNA condensation. Increased condensation may lead to slower embryo development and higher embryonic death. Other reports suggest damage may involve nuclear cytoskeletal changes that allows early de-condensation and male pronucleus formation before fertilization. All changes may result in lowered fertility. Certain assays such as the Sperm chromatin structure assay (SCSA) are beneficial for identifying the potential for sperm damage and predictive boar fertility. The DNA damage that may result from ice crystal formation or oxidative damage, is irreparable since sperm have no DNA repair mechanism. However, DNA damaged sperm can fertilize eggs but can lead to increased embryo loss. Frozen boar sperm has been associated with high levels of DNA damage independent of normal semen fertility measures. Future efforts will focus on cryoprotectants, antioxidants, and the seminal plasma effects on DNA integrity. Boar sperm stressors during processing Methods to improve sperm and egg survival during freeze-thawing can be increased by pressurized stress conditioning (Pribenszky et al., 2011). Pre-conditioning of sperm for survival was originally noted by cold incubation before freezing. Stress preconditioning has been noted in many organisms and is commonly known as adaptation. The process uses gradual pressurization of boar sperm over 2 min with MPa with holding at 90 min followed by gradual decompression before the freezing procedure. In tests, multiple ejaculates were collected and extended and cooled to RT and then split into pressurized and non-pressurized aliquots. Semen was then used for AI, or stored in extended form for 12 d or frozen in 0.5 cc straws. Pregnancy rate did not differ among treatments (74%) but litter size was increased in gilts (not sows) and motility of sperm increased at 5 days of storage. The interpretation of data leads to the concept that the spermadhesin proteins are altered during pressurization and that some of these may also be involved in the energy ATP pathway involving oxidative phosphorylation and the generation of the reactive oxygen intermediates. Since boar sperm undergo stressors during processing and freezing, modification of boar diet could be used to alleviate this effect (Radomil et al., 2011). Damage from freezing and thawing can result in sperm cell cold shock, intra-cellular ice crystal formation that can damage to mitochondria, membranes, acrosome, and tail, as well as the creation of reactive oxygen intermediates. Oxidation may have greater negative effects on motility, acrosome integrity, and membrane reorganization from lipid peroxidation more so than on viability. The sperm membrane of the boar is also more likely to be affected as it is higher in poly unsaturated fatty acids (PUFA). The loss of selected lipids from the membrane during the cryopreservation process has been observed. It is thought that replacement of these could improve post thaw survival. Antioxidants such as vitamin E can reduce the oxidative damage to PUFU. Diets are usually low in the ratio of long chain Omega 3 : Omega 6 PUFA. Inclusion of higher ratios of Omega 3 PUFU from flaxseed in the diet of three boars for 9 weeks was performed with semen collected twice/week. Cooled semen was evaluated and results indicated that diet increased normal sperm, motility, and viability during storage but did not impact capacitation. There may be some potential to alter sperm fertility with diet. Sperm Evaluation systems Sperm assessment methods (Waberski et al., 2011) can be used to evaluate the effects of processing and storage on sperm. Sperm can undergo lethal changes as evidenced by loss of motility or viability or sub-lethal changes not clearly identified by standard methods. Detection of sub-lethal changes in sperm cells may be important and can be facilitated using computerized analysis and sperm sorting systems. These procedures require multiple fluorescent stains that can reveal more subtle damage effects of storage on boar sperm. 4

5 CASA systems are commonly used today and are important in boar sperm production (Feitsma et al., 2011). In some studies CASA measures have been associated with fertility and some systems can assess concentration, motility, viability and morphology. The machines can count large numbers of cells can be evaluated for their accuracy and repeatability using standard lab methods. The choice to purchase a machine should depend upon the cost, variation in current lab measures and skill of personnel. Loss of accuracy with CASA is most often associated with improper sample mixing, dilution, loading, counting chamber toxicity, chamber depth, or system settings. Swine AI Since pig sperm can survive in the reproductive tract for ~24 h following AI, and survival for frozenthawed sperm is much shorter, multiple inseminations are commonly performed to compensate for variation in time of ovulation. A new approach to extend the in vivo life-span of boar sperm in the female is tested with use of a controlled sperm release system by encapsulation of sperm in a matrix (Faustini, 2011). This approach used barium alginate matrix to encapsulate boar sperm which allows molecules to enter and exit sperm. Trials indicated that sperm could be maintained in fertile states longer and at higher levels compared to no-treated sperm while in storage. At 37 C, higher sperm viability is observed as well. In AI experiments, high farrowing rates and good litter sizes result with a single AI using 5 billion treated sperm. Future work will focus on cryopreservation of the sperm capsules for use with even lower sperm numbers. Boar Fertility Markers Semen analyses can be associated with fertility but is most often not predictive of boar fertility. Functional sperm tests and molecular tests can be associated with one or more sperm traits related to fertility. These can be compensable in that the more sperm inseminated the higher the fertility or noncompensable where more sperm does not improve fertility. Most of the lab tests have had issues with repeatability which have limited their application. In some species, proteins such as osteopontin and in boars, the seminal plasma proteins (sperm adhesins) have been associated with higher or lower fertility. The adhesin protein, PSP 1 is the primary SP protein in the pig. It is involved in prevention of capacitation and the acrosome reaction, and may also have immune regulator function as well. Fertility association with the SP proteins may only be evident when lower numbers of sperm are used. The complexity in using these proteins as markers for fertility may depend upon how they are detected, at what stage of development, and how they are used to predict a function related to fertility. Semen analysis and fertility AI companies focus boar semen fertility to help establish guidelines for use and rejection of ejaculates and to explain fertility (Broekhuijse et al., 2011). In the Netherlands, data was shared from 1800 boars and 132,000 ejaculates used to mate 4.3 million sows at 750 different farms with a result of 8.6 million farrowing records. The semen was produced at regulated AI stations using SOPs and analyzed using CASA. Ejaculates contained on average 84 billion cells and 35 doses/boar/ejaculate resulted in AI doses containing 1.5 billion motile sperm. Females received 1.6 AI doses depending upon days standing and were checked every 12 h for estrus. Boar line and boar effected fertility. Morphology also had an effect and reducing abnormal sperm from 30 to 20% increased farrowing rate by 0.07% and LS by 0.08 total born. There was no effect of day of storage up to 5 days on fertility. CASA use removed lab technician effects from motility on fertility and was able to show a small but significant effect of CASA motility on fertility. 5

6 Risk of disease from semen AI allows rapid dissemination of valuable genes but can also spread disease with the same efficiency (Althouse and Rossow, 2011). The most common route for disease entry into farms or studs is by new animal introductions. In boar studs this can be quite high due to annual replacement rates of %. Other routes of disease entry can occur from aerosolization, fomites, people, insects, vectors, birds and mammals. There are only a few infectious bacterial diseases of concern to swine. Brucellosis is a major disease in certain parts of the world. Upon infection, the organism is localized to the reproductive tract and can be spread by the boar and semen. Tests are available for diagnosis for both. Chlamydia is disease is a widespread disease that originates from an intestinal microbe that can be localized to the urogenital tract. In many cases the disease may be asymptomatic but transmission by semen is possible. Tests are available but limited information on transmission and control are available. Leptospirosis is a common disease of swine that can cause reproductive failure. These bacteria can localize in the reproductive tract and carrier and shedding states can exist in fluids such as semen. Control can include vaccination and regulation of access and source of water. Some types of bacteria may be normally present with the boar and can contaminate a semen sample during collection. Hygiene and antibiotics can be used to control this occurrence. Unlike bacteria, there are numerous viruses of global importance that can infect swine and which can be spread by semen. These include PCV2 which is present on most farms and can cause problems. Testing in not common and vaccines available. PRRS is an important disease of swine and the agent can be transmitted by boars and semen. The virus can be shed in semen for months. The mutable forms of the virus prevent vaccination from always being effective in infection, re-infection or shedding. Boar and semen can be tested by PCR. Parvovirus is highly contagious through oral-nasal routes and can colonize the reproductive tract with subsequent spread via semen to cause reproductive losses. Vaccination or immunity can be used to prevent disease. Pseudorabies is an important global disease spread primarily by animal contact. The disease can colonize the reproductive tract and can be spread via semen. Screening boars and samples is practiced to prevent transmission. Vaccines are available as well. Classical swine fever or hog cholera and Foot and mouth disease are highly contagious diseases endemic to regions around the globe. Tests and vaccines are used for control. Japanese encephalitis is a disease of concern in Asia and is spread by mosquitos. Infection results with orchitis and virus shedding in semen. Tests and vaccines are available for control. Sperm Storage in the female reproductive tract Sperm storage in the female reproductive tract following insemination appears to be related to species evolution through anatomical adaptation. The story of sperm storage may be more complex than a storage space for sperm until the time of release to allow fertilization when ovulation occurs. A review of this process (Holt, 2011) suggests more complexity as not all sperm are allowed access to the site of fertilization or storage. Species differences exist in storage location which may occur near the oviduct in the pig, the ovary in some fishes, a blind sac with sperm storage tubules (SSTs) near the utero vaginal junction in birds, and in the oviduct of sharks, reptiles and cloaca of some amphibians. In most mammals, storage is used to control insemination to ovulation intervals of hours to several days. However some unique species of mammals such as bats have functional sperm reservoirs that last several months since mating occurs in fall and ovulation occurs in spring. Some fish and reptiles allow sperm storage of over year. Some females have been shown to store semen from multiple males at a time and then have offspring biased toward one of the males or toward one of the sexes. Sperm storage appears an evolutionary adaption with a phylogenetic relationship among groups of vertebrates containing birds, amphibians, and reptiles with reservoirs lasting from one breeding season to the next. The mechanism regulating extended sperm life in mammals although relatively short compared to other 6

7 species may not only be related to unique anatomical structures, but also depend upon gene expression and synthesis of proteins resulting from sperm binding to female epithelial cells in the reproductive tract which then extends sperm viability and appears common across several species. The genes expressed and identified appear related to heat shock proteins in some species and in others factors related to immunologic regulation such as tumor necrosis factor and transforming growth factor. In birds, it appears that sperm can move from a low reservoir near the cloaca, to a higher one near the oviduct and appears dependent upon sperm surface interaction with the epithelial cells at a site. But the epithelial cell type does not seem to be specific as sperm exposure to any epithelial cell type cells results in extended life and binding in vitro. Further, sperm become immobile upon epithelial cell binding when calcium and temperature are at body temperature, but remain motile when examined at room temperature. This observation for birds is similar to that observed for the pig and the rabbit. Since many species have common cellular mechanisms to bind and react to sperm the likely explanation for much longer sperm survival favors the unique SSTs. As such, inserting sperm in temporary artificial tubules could allow extended fertilization periods in swine. Maternal communication with embryos Much more of the apparent reproductive failure to establish pregnancy may be due to excessive embryo loss than fertilization failure. Eggs that are fertilized may be lost as embryos and pregnancies failed to become recognized prior to implantation. Excessive embryo loss and not fertilization failure may help explain elusive reproductive failure in many species but is not detected by extended menstrual cycles of women or reproductive cycles of livestock (Fazeli, 2011). Early maternal communication with embryos may occur much earlier than previously thought. Compared to unfertilized eggs, embryos induce differential RNA expression within different oviduct cells in the same animal. This work clearly shows that sperm and eggs can act locally to influence gene expression in the oviduct cells while hormones affect these cells systemically. The proteins produced by the epithelial oviduct cells have effects on gamete maturation, viability and function. This suggests that the maternal tract responds to the presence of sperm, eggs or embryos to modulate its ability to allow sperm attachment, survival and release, egg fertilization, and embryo development and survival. New modeling systems are under development to study this complex process. Towards efficient use of boar sperm Approaches toward using few sperm is important to extend use of valued sires and to address the limitations to numbers of sperm from frozen and sex sorted semen. Procedures such as intrauterine insemination (IUI) and deep uterine insemination (DUI) can help to address these shortfalls (Roca et al., 2011). When using liquid semen, IUI can lower numbers required to 1.0 billion sperm to meet fertility expectations. With frozen sperm, while IUI has not helped improved fertility, conventional AI (CAI) has been successful by itself with resulting farrowing rates >80% and total born from in some studies. However, with frozen sperm, the requirement for high numbers of sperm (5-6 billion) in 2 to 3 inseminations points to problems with sperm damage and survival. On the other hand, DUI results seem to be more encouraging with frozen sperm use and fertility expectations can be met when using only 1 billion frozen sperm. Sex-sorted sperm also holds great potential for the industry but these sperm have a limited fertile lifespan and upon freezing are even more fragile than non-sorted frozen sperm. Timed AI with ovulation induction with use of DUI may be required for success when using very few sperm in frozen form. 7

8 References Althouse, G. C., and K. Rossow The Potential Risk of Infectious Disease Dissemination Via Artificial Insemination in Swine. Reproduction in Domestic Animals 46: Bathgate, R Antioxidant Mechanisms and their Benefit on Post-thaw Boar Sperm Quality. Reproduction in Domestic Animals 46: Broekhuijse, M., H. Feitsma, and B. M. Gadella Field Data Analysis of Boar Semen Quality. Reproduction in Domestic Animals 46: Faustini, M New Aspects of Boar Sperm Encapsulation. Reproduction in Domestic Animals 46: Fazeli, A Maternal Communication with Gametes and Embryo: A Personal Opinion. Reproduction in Domestic Animals 46: Feitsma, H., M. Broekhuijse, and B. M. Gadella Do CASA Systems Satisfy Consumers Demands? A Critical Analysis. Reproduction in Domestic Animals 46: Fraser, L., J. Strzeżek, and W. Kordan Effect of Freezing on Sperm Nuclear DNA. Reproduction in Domestic Animals 46: Holt, W. V Mechanisms of Sperm Storage in the Female Reproductive Tract: an Interspecies Comparison. Reproduction in Domestic Animals 46: Knox, R. V The Current Value of Frozen Thawed Boar Semen for Commercial Companies. Reproduction in Domestic Animals 46: 4-6. Leahy, T., and B. M. Gadella Capacitation and Capacitation-like Sperm Surface Changes Induced by Handling Boar Semen. Reproduction in Domestic Animals 46: Morrell, J. M., and M. Wallgren Colloid Centrifugation of Boar Semen. Reproduction in Domestic Animals 46: Pribenszky, C., A. Horváth, L. Végh, S. Y. Huang, Y. H. Kuo, and O. Szenci Stress Preconditioning of Boar Spermatozoa: A New Approach to Enhance Semen Quality*. Reproduction in Domestic Animals 46: Radomil, L., M. J. Pettitt, K. M. Merkies, K. D. Hickey, and M. M. Buhr Stress and Dietary Factors Modify Boar Sperm for Processing. Reproduction in Domestic Animals 46: Riesenbeck, A Review on International Trade with Boar Semen. Reproduction in Domestic Animals 46: 1-3. Roca, J., I. Parrilla, H. Rodriguez-Martinez, M. A. Gil, C. Cuello, J. M. Vazquez, and E. A. Martinez Approaches Towards Efficient Use of Boar Semen in the Pig Industry. Reproduction in Domestic Animals 46: Safranski, T. J., J. J. Ford, G. A. Rohrer, and H. D. Guthrie Plenary Contribution to International Conference on Boar Semen Preservation Genetic Selection for Freezability and its Controversy with Selection for Performance. Reproduction in Domestic Animals 46: Waberski, D., H. Henning, and A. M. Petrunkina Assessment of storage effects in liquid preserved boar semen. Reproduction in Domestic Animals 46: