Artificial insemination (AI) was the first great biotechnology applied to improve reproduction and genetics of farm animals. Next developed procedure to the cryopreservate semen made great step towards different possibilities almost unlimited preservation of pathogen free quality genotypes semen, its usage in different time and locations, making transport easier and manipulative fees more acceptable. Limitations of the technique consider biological traits of semen and difficulties to reproduce them. The knowledge gained from the AI and the cryopreservation experience was extremely helpful in stepwise development of each successive reproductive technology, such as superovulation, embryo transfer, and, eventually, cloning

Hristov, Slavča

Petrujkić, Tihomir

Relić, Renata

Stanković, Branislav

English

AgroSpace

139 „SAVREMENA POLJOPRIVREDA” VOL. 54, 1–2 (2005) STR. 139–144, NOVI SAD FREEZING THE BOARS SEMEN: SOME LIMITATIONS  AND PERSPECTIVES* STANKOVIĆ B. 1, PETRUJKIĆ T.2, HRISTOV S.1, RELIĆ RENATA1  ABSTRACT: Artificial insemination (AI) was the first great biotechnology applied to improve reproduction and genetics of farm animals. Next developed procedure, to the cryopreservate semen made great step towards different possibilities: almost unlimited preservation of pathogen free quality genotypes semen, its usage in different time and locations, making transport easier and manipulative fees more acceptabile. Limitations of the technique consider biological traits of semen and difficulties to reproduce them. The knowledge gained from the AI and the cryopreservation experience was extremely helpful in stepwise development of each successive reproductive technology, such as superovulation, embryo transfer, and, eventually, cloning.  Key words: boar semen, cryopreservation, extenders, perspectives. BEGINNINGS OF AI, FROZEN/THAWED SEMEN USAGE  BEGINNINGS AND PROCEDURE The artificial insemination (AI) of swine was initiated by Ivanow in Russia (Ivanow, 1907; 1922) and continued using artificial vaginas or a gloved hand (McKenzie, 1931; Ito et al., 1948; Polge, 1956). Extenders were based on glucose solutions of Na-K-tartarate or Na2SO4 and peptone keeping the concentration of electrolytes low, including cooled semen (Ito et al. 1948). Although AI was succesfully performed by Ito et al. (1948) in Japan, it took time before it became common method in swine reproduction.  While breakthroughs were made on sire selection, a great achievement was reported from England (Polge et al., 1949): successfully freezing chicken sperm using glycerol. Glycerol soon was found to be useful for bull sperm, but not for the boars. Accidental discovery of Polge, (1968), focused on sugars as cryoprotectants, showed that glycerol and albumin obtained hopeful results. The basic medium used by Polge was the original yolk-citrate extender (Salisbury et al., 1941) and glycerol. O’Dell and Alm-quist (1957) developed milk-glycerol as a cryopreserving medium to bull sperm, as well as Tris-buffered egg yolk-glycerol (Davis et al., 1963; Foote, 1998), which became the most commonly worldwide used cryopreservation medium for of bull semen and several   1 Mr Stanković Branislav, teaching assistant, dr Hristov Slavča, associated professor, DVM Relić Renata, teaching assistant, Faculty of Agriculture, Belgrade - Zemun 2 Dr Petrujkić Tihomir, teaching professor, Faculty of veterinary medicine, Belgrade *This paper is financed by Ministry of Science of Serbia, BTN 7103B 140 other species (Iritani, 1980), but not boars. It was shown its toxicity to boar semen, in-creasing permeability of cell membrane (Larsson, 1978). It took many years of work to freeze boar semen, following the successful use of frozen bull sperm (Nishikawa, 1964; Iritani, 1980; Johnson and Rath, 1991; Foote, 1999; Johnson and Guthrie, 2000). Polge et al. (1970) performed fhe first successful deep frozen boar semen AI. Crabo et al. (1971) developed succesfull thawing procedure with seminal plasma as extender, but Beltsville method, developed by Pursel and Johnson (BTS - Betsville Thawing Solution, 1975) was more applicable, as it is today, with few modifications, mostly considering type of extender, with these phases: 1. equilibration on 20 oC during two hours, 2. separation of seminal plasma by centrifuge, 3. addition of BF5 extender and cooling to 5 oC, d. addition BF5 and glycerol, 4. freezing semen (peletts/straws) to – 79 oC, and – 196 oC. Until 1991, new boar semen extenders compo-sition was presented, but now it is not revealed because of the competition among manu-facturing companies, competing for a huge market for long-term (7 - 10 days) boar se-men extenders. Reasons for developing new long-term boar semen extenders relate to: more favorable work hours in the boar stud, reduced transportation cost of delivering semen to the farm, testing of semen for Porcine Reproductive and Respiratory Syndrome virus before use, and a longer period for using the semen with satisfactory reproductive performance (Levis, 2002). First of extenders of the denominated ones long play it was the Zorlesco (Gottardi ET al., 1980), with TRIS pH regulator, bovine serum albumin (BSA) and cistein, stabilizing membranes and inhibiting the qualification process (John-son et al., 2000), without satisfactory results, as well as Moretti’s (1981) Modena exten-der with more glucose and no BSA (Johnson et al., 1988; Laforest and Allard, 1996). In 1990 was presented Androhep extender, containing HEPES as pH regulator and BSA, which keep osmotic pressure. This extender was accepted as extender of long conserva-tion, followed by Acromax, X-Cell, Androhep Extra, Vital, SpermAid, Mulberry III, etc. It must be emphasized that average egg lactose-yolk extender is more frequently used and described by Pursel and Johnson (1975) denominated BF-5, in whose com-position it includes glucose, yolk of egg and Tris as pH regulator. LIMITATIONS Few serious problems occurred in establishing of frozen/thawed semen AI techni-que. Cryopreserving boar semen results in an extreme reduction in fertilizing ability (Almlid and Johnson, 1987; Parks and Graham, 1992), possibly due to damage to sperm membranes (Watson and Plummer, 1985; Maxwell and Johnson, 1997). Spermatozoa must be concentrated before freezing. Plasma and acrosomal membranes, their structural and functional integrity, influence sperm shape and volume motility, so energy produc-tion, permeability, capacitation and acrosome reaction and interaction with oocytes are very important to sperm. Cryopreservation (dilution, cooling, freezing/thawing) alters selective permeability of sperm membranes, reduces motility and energy production and changes membrane dynamic behavior (He et al., 2001). Membrane lipid composition is intimately involved in the degree and nature of the damage caused by cryopreservation (Quinn, 1989). Sperm from different parts of the epididymis have different sensitivity to cold shock, correlated to changes in lipid composition during sperm maturation from 141 caput to cauda epididymis (Bwanga,1991). Buffer system of extender, simmilar to same system of the seminal plasma, plays important roll in frozen semen viability. It was demonstrated that bicarbonate and calcium play crucial roles in the capacitation process in pig sperm. Specific tyrosine-phosphoprotein appears during capacitation in a calcium-dependent manner, but its presence is not a prerequisite for capacitation of pig sperm (Tardif et al., 2003)  Just 10% of tested boars population provides needed quality of semen in respect of spermatozoa viability, survival and fertilization ability after thawing. Pregnancy rates and litter sizes are reduced with cryopreserved semen, so fresh or extended liquid semen is used for about 99% of AI in swine. Frozen semen is limited to use in special breeding programs. Low conception rate and litter size obtained by thawed semen insemination is not satisfying (Johnson et al. 1980), as many semen additives to improve fertility of frozen semen have been tested with minimal success (Foote, 1999), but data were provided for a peptide that increased fertility added to frozen/thawed semen (Amann et al., 1999). PERSPECTIVES New semen extenders generated on long-term viability of semen, farrowing rate and litter size data are presented by the company with a business interest, with aggressive advertisement and marketing, not peer reviewed or published in scientific journals. Only few boar studs/pork production enterprises compare extenders scientifically. The knowledge gained from the AI and cryopreservation was extremely helpful in stepwise development of various technologies, such as superovulation, embryotransfer, and, eventually, cloning. Sexing of boar sperm is possible but slow for commercial use. Equipment is permanently developed, such as spermatozoa sorters (Foote, 2001). Design of new extenders and knowing more the spermatozoa metabolism are necessary, as well as using new systems of evaluating and optimizing the components of the extender (Pettit et al., 1999). Diverse additives, like alkil-glycerol must be evaluated in respect of the viability of the spermatozoa (Cheminade et al., 2002), cold shock (Zeng and Terada, 2001), improving the spermatozoa transport synchronized with the ovulation and fertilization (Waberski, 1997; Kemp and Soede, 1997). Problem of the thermal shock must be resolved, so equipment the elasticity effect on the spermatozoa survival and fertility results is evaluating (Althouse et al., 1998; Paulenz et al., 2000). Levis (2000) suggests that aspects of the artificial insemination, such as the individual diffe-rences, are not studied well, since all the males do not interact of equal way with used means (Weitze, 1990). Development of new systems like deep the intrauterine inse-mination and reduced number of spermatozoa by dose and the volume of insemination (from three to billion spermatozoa per milliliter of extended semen) requires new conditions of conservation. Of course, it will be necessary to study the best extender and freezing technique (Rath, 2002). CONCLUSIONS In order to make usage of fozen/thawed boar semen common, further investigations must take place: 142 • Diverse extenders and additives must be devoped and evaluated in respect of the viability of the semen; • Problem of the thermal shock must be resolved, as well as development of new technicques (deep intrauterine insemination, e.g.), with reduced number of spermatozoa by dose and the volume of insemination and adaptation of extenders to the individual differences. Untill that happen, freezing of boar semen will be procedure limited in possibilities, time and space. REFERENCES CITED ALMLID T, JOHNSON LA 1987. Fertility evaluation of the straw freezing technique for boar semen under practical artificial insemination conditions. Zuchthygiene (Berl); 22:193–202.  ALTHOUSE G. C., WILSON M. E., KUSTER C., PARSLEY M., 1998. 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Freezing the boars semen: Some limitations and perspectives

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Freezing the boars semen: Some limitations and perspectives

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