RESEARCHES REGARDING BOAR SEMEN CRYOPRESERVATION

UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINRY MEDICINE CLUJ-NAPOCA DOCTORAL SCHOOL Faculty of Veterinary Medicine SOTOC OCTAV CORNELIU SORIN SUMMARY OF THE PHD THESIS RESEARCHES REGARDING BOAR SEMEN CRYOPRESERVATION Scientific coordinator, Prof. Univ. Dr. IOAN GROZA Cluj-Napoca 2011

INTRODUCTION The onset of boar semen freezing activity was recorded in 1956, when Polge has successfully realized this procedure for bull sperm. Research carried out later on boar sperm showed an unexpected fact there is a difference between the cooling and freezing boar and bull sperm. Evidence indicates that there are differences in the sensitivity of mammalian sperm to cryopreservation, so it is almost impossible to extrapolate techniques from one species to another. Efforts to freezing boar semen were successful in 1970, when fertilized oocytes obtained after insemination with semen frozen and then thawed (Woelders H. et al., 2005). This success was followed by others, the process of insemination was this time intracervical (Crabo et al., 1971, Graham et al., 1971, Pursel et al., 1971). The general conclusion of experts in the field was that frozen boar semen has a low fertility, even if increasing the number of sperm per dose (Johnson, 1985.1998, Johnson et al., 2000). The causes which reduce performance of boar semen frozen/thawed is assumed to be due to: reduction of viability and motility of sperm frozen/thawed; increased sensitivity to cryopreservation of boar sperm; staff experience. PERSONAL RESEARCH Purpose of the paper Given the importance of the biotechnology, research were focused to several directions: improving the methods for assessing sperm parameters by introducing morphocytometric analysis to supplement the existing methodology based strictly on morphological aspects; determining the freezing efficiency by using more standardized medium for pre- freezing dilution of boar semen; development and testing original medias for processing and freezing of boar semen; determining qualitative changes occurring during freezing, especially in terms of morphocytometric exam and temporal evolution post-thawing; estimate semen pregnancy rate obtained by artificial insemination technique; determining the quality of frozen semen with standard freezing media and frozen semen using the original media, through artificial insemination. I

BOAR EVALUATION AND SELECTION REGARDIN SPERM CRYOPRESERVATION PURPOSE OF RESEARCH The aim of this chapter is the assessment of semen quality and it s suitability to the processing and freezing methods. Semen was examined taking into account age donors and monitoring the differences between parameters according to age. Also we proposed the improvement of swine sperm morphological assessment by introducing the morphocytometric evaluation of length and width sperm head. MATERIAL AND METHOD The research was carried out between February 2007 - November 2010 in the Clinic of Reproduction, Obstetrics and Gynecology Faculty of Veterinary Medicine of Cluj-Napoca in collaboration with a private firm in the county of Arad. Sperm samples from 20 boars, Large White and Landrace, were analyzed. Boars were grouped according to age into two experimental batches: batch I (10 boars), 5 each of every breed, recently introduced for breeding, aged between 8 months to 1.5 years; batch II (10 boars), 5 each of every breed, using intensive breeding, aged between 2 and 3.5 years. Boars were kept in similar conditions, being housed in individual boxes, arranged in the same shelter. The animals were fed with the same type of feed, the amount varying with the weight of each. Semen collection was performed in the unit of boars origin with team members of the Department of Reproduction, Obstetrics and Veterinary Gynecology, assisted by staff unit. For operational and commercial reasons, semen collection was performed three times (every 7 days) realizing each time semen evaluation. Mention that semen collection has been made on the same day for all boars. Semen was collected by manual method after the jump boar on the mannequin. Semen evaluation involved an macroscopic analysis completed by a microscopic examination and sperm morphocytometry. Macroscopic analysis determined the following parameters: volume, odor, color. Microscopic exam determined concentration (with SpermaCue device), mobility, density, viability (eosin 5% stain) and description of morphology (physiological or pathological) of sperm (Spermac stain). Morphocytometry was performed using optical microscopy with inversion and computer, using the software Motic Images Plus located in Spermolab laboratory of the Department of Reproduction, Obstetrics and Veterinary Gynecology. n terms of technique, this involved computerized measurement of the length and width sperm head. II

RESULTS AND DISCUSSION Assessment of semen parameters focused on macro-and microscopic determination specific for each boar, and the results were included in andrological sheet prepared individually. Depending on the results the semen samples have been accepted or rejected from processing regarding cryopreservation and and artificial insemination. In batch I the volume of semen ranged from 52-160 ml in Large White boars and between 62-184 ml Landrace breed. Recorded values of sperm concentration ranged from 128-343 x 10 6 sperm/ml in Large White and between 149-444 x 10 6 sperm/ml in Landrace boars. The percentage of mobile sperm ranged from 55-80% for Large White boars and between 55-87% for Landrace breed. Analyzing the values of sperm viability in both breeds of pigs included in the experiment shows that this parameter ranged from 65-90%. In terms of morphological examination, normal sperm were found in the proportion of 85-95% for the two breeds of boars. Primary abnormalities ranged from 3 to 4.2% in Large White and between 2.6 to 4% in Landrace breed, and the secondary abnormalities between 7.6-8% in Large White and between 5 to 7.4% in Landrace boars. Regarding sperm head length there is a slight difference in favor of Landrace boars (6.96 to 8.75 µm) to the Large White (6.50 to 8.60 µm). Sperm head width ranged from 2.80 to 3.94 µm in Large White boars and between 3 to 4.10 µm in Landrace boars. In batch II the volume of semen ranged from 63-230 ml in Large White boars and boars between 74-250 ml in Landrace. Sperm concentration varied in large limits, is rarely the reason for poor semen quality and often exceeding the value of 300 x 10 6 sperm / ml. Motility before conservation was between 70-95% in Large White and between 75-95% in Landrace breed. Sperm viability ranged from 74-95% for Large White boars and between 78-95% for the Landrace breed. The average percentage of morphologically normal sperm detected with Spermac method ranged from 89.8 to 94% for both breeds of pigs included in the experiment. The morphocytometric study of sperm head were found minor differences between length and width, average for the parameters studied fits within limits considered normal for swine species. Summary of the average values of sperm parameters No. C Vi Morphology (%) Morphocytometry (µm) Breed Vol (ml) batch (x 10 6 M (%) spz/ml) (%) N Pa Sa l w Batch L.W. 117.66 236.33 67.46 82 88.33 3.86 7.80 6.50-8.60 2.80-3.94 I L 120.40 290.73 71.66 83.66 90.33 3.53 6.13 6.96-8.75 3.00-4.10 Batch L.W. 174.20 349.46 85.20 88.80 91.46 3.20 5.33 7.26-8.74 3.15-4.23 II L 182.40 405.40 88,73 90.60 92.66 2.73 4.60 7.79-8.84 3.84-4.37 L.W. Large White; L Landrace; Vol. volume; C- concentration; M motility; Vi. viability; N normal spermatozoa; Pa Primary abnormalities; Sa secondary abnormalities; l- length; w width; III

BOAR SEMEN CRYOPRESERVATION AND ESTABLISHMENT OF IT S EFFICIENCY BY LABORATORY METHODS PURPOSE OF RESEARCH Research carried out in this chapter were designed to respond to several purposes as follows: setting freezing efficiency by using more standardized medium for dilution of boar semen; development and testing original media for freezing boar semen; estimation of changes occurring during freezing, in terms of sperm mobility, viability and morphology; morphocytometric exam of sperm with the estimation of their size variation during the process of cryopreservation; use and temporal evolution expression of the quality indicators of postthawing semen, depending on the extender used for freezing. MATERIAL AND METHOD The research was carried out between 2007-2010 on a number of 35 semen samples, representing 58.33% of the total collected. Were accepted for processing those ejaculates that showed more than 75% motile and normal sperms. Samples that have been subject to research were collected as follows: at the first harvest were obtained 13 samples of semen; at the second harvest were obtained 12 samples of semen; at the third harvest were obtained 11 samples of semen. Dilution of boar semen Immediately after determining the concentration, semen dilution was performed, as a preceding step for cryopreservation To dilute semen samples were tested for efficiency three commercial dilution media: BTS medium allows preservation of semen for 2 days; MERCK III medium allows preservation of semen for 4 days; ANDROHEP Plus medium allows preservation of semen for 7 days. IV

a) b) c) Commercial media tested:: a) BTS ; b) MERCK III ; c) ANDROHEP Plus The composition of the dilution media was modified, eesulting for each medium two experimental variants. Cryopreservation of boar semen Semen freezing protocol followed the steps recommended by Pena et al., 2003, Bianchi et al., 2008, with certain amendments proposed by the staff of the Department of Veterinary Reproduction, Obstetrics and Gynecology and the PhD student, taking into consideration the following protocol: diluted semen in 1:2 ratio with dilution media (A medium), prewarmed at 32.5 0 C, was aliquoted into 4 conical Falcon tubes and maintained at 22 0 C for 1 hour; the tubes were maintained at 15 C for 3h inside the centrifuge; the semen was centrifuged at 900g (3500 rpm) for 10 minutes at 15 C; Semen centrifugation after centrifugation the supernatant was discarded while the sediment was resuspended with 80 ml β-lactose and 20 ml egg yolk (B medium); temperature decreased gradually with 0.2 0 C/minute, from 15 to 5 0 C; the third dilution was done with 89.5 ml B medium plus 9 ml glycerol 3% (C medium), kept at 4 C; V

the 0.5 ml straws were filled with diluted semen at 4 0 C using the MiniTube semi-automatic Filling&Sealling Machine (MiniTube); freezing was performed by placing the straws 4-5 cm above liquid nitrogen vapor on a metal support, for 20 minutes; storage temperature decreased from 5 to -120 0 C, with a decline rate of 30 0 C/minut; the final step consisted on tranfgerring the straws from the metallic holder to the liquid nitrogen container. Thawing of boar semen Thawing was performed by plunging the straws into a bath water at at 38 0 C for 20 seconds (Roca et al., 2006). After thawing, they following sperm parameters were microscopically appreciated: mobility, viability, morphological and morphocytometric exams. Semen microscopic evaluation RESULTS AND DISCUSSION Thawing semen protocol in straws was followed by appreciation in terms of mobility, viability, morphology and sperm morphocytometry. Thawing results were recorded in individual sheets of thawing. Testing the BTS media were registered the following results: evaluation of freezing media based on BTS allowed the maintenance of sperm mobility between 25-26% after freezing; sperm viability ranged from 28 to 31%; percentage of dead sperm ranged from 69-72%; percentage of normal sperm morphology was recorded between 86 to 91.5%; abnormalities were found in percentage of 8.50 to 14%. Testing the MERCKIII media were registered the following results: evaluation evaluation of freezing media based on MERCKIII allowed to obtain a sperm mobility between 25 to 28.1%; sperm viability ranged from 30-34% ; VI

percentage of dead sperm ranged from 66-70%; freezing boar semen allowed to obtain good percentage of normal sperm between 89-93%; abnormalities were found in percentage of 7-11%. Testing the ANDROHEP Plus media were registered the following results: freezing media, based on ANDROHEP Plus has allowed maintaining the mobility of sperm from 26.6 to 28.1%; sperm viability in ANDROHEP Plus mediums ranged from 31-34%; percentage of dead sperm ranged from 66-69%; freezing boar semen allowed to obtain good percentage of normal sperm between 89-92%; abnormalities were found in percentage of 8-11%. Morphocytometric analysis revealed normal values of length and width of sperm head with discrete reduction after freezing. Morphocytometric parameters determination VII

CRYOPRESERVATION EFFICIENCY ASSESSMENT BY ARTIFICIAL INSEMINATION PURPOSE OF RESEARCH Apart from the laboratory semen evaluations, such as assessments of mobility, vitality, normal spermatozoa percentage, along with their temporal maintenance and persistence, the pregnancy rate represents a supplementary but also the most relevant freezing and frozen semen quality evaluation procedure. Therefore, sows artificial insemination with semen obtained by using different freezing media, was performed for the following purposes: achieving hormonal treatment for induction and estrus synchronization in sows; global comparison of pregnancy rates for insemination with frozen or diluted semen; establishing the suitability of 3 original media (BTS 1, MIII 2, A 2 ) for artificial insemination; evaluation of pregnancy rate of sows inseminated with semen frozen in their environments. MATERIAL AND METHOD The research was carried out between 2007-2010 on an effective of 180 sows of different ages and races belonging to private farms in Arad county, aged 1-4 years, half-breeds Large White and Landrace. Sows in evidence, were divided into four batches of 45 sows each. Hormonal therapy and sperm category used for artificial insemination Entry Batch No. sows Hormonal treatment Sperm category 1 Batch I 45 Physiological estrus Diluted sperm 2 Batch II 45 Physiological estrus Frozen sperm 3 Batch III 45 Regumate individually feed Diluted sperm 4 Batch IV 45 Regumate individually feed Frozen sperm VIII

Aspects during artificial insemination RESULTS AND DISCUSSION The return percentage was positive in batches I and II (0) compared with groups (III and IV) subjected to estrus synchronization were the values recoded were between 6.66 and 11.11%. Installation of gestation in batches artificially inseminated with diluted semen was 91.11% in group I and 80% in group III. At the batches artificially inseminated with frozen semen pregnancy was confirmed in 75.55 percent in group II and 68.88 in group IV. When using frozen semen highest pregnancy rate was achieved with the A 2 medium (26.66%), followed by the other two batches where the values ranged between 22.22-24.44%. The average number of born piglets was higher in groups that artificial insemination was performed with diluted semen (group I 11.97 piglets/sow, group III - 11.15 piglets/sow) compared with groups who used semen frozen (group II 9.93 piglets/sow, group IV 9.70 piglets/sow). Regarding the number of weaned piglets, the values recorded in groups I and III were between 9.65 and 10 weaned piglets /sow and in batches II and IV between 8.15 and 8.51 weaned piglets /sow. Results obtained after artificial insemination PHYSIOLOGICAL DIAGNOSIS ESTRUS SYNCHRONIZATION Batch I Batch II Batch III Batch IV Estrus 45 45 41 42 Anestrus 0 0 4 3 G after first cycle 43 36 38 32 G after second cycle 2 4 3 5 Nonpregnant 0 5 4 5 Born piglets 11.97 9.93 11.15 9.70 Weaned piglets 10 8.51 9.65 8.15 GENERAL CONCLUSIONS IX

Following research carried out between February 2007 - November 2010, on a total number of 20 boars of Large White and Landrace breeds, aged between 8 months and 3.5 years, clinically healthy, were formulated the following conclusions: 1. in batch I, the volume of semen ranged from 52-160 ml in Large White boars and between 62-184 ml Landrace breed; 2. values recorded in batch I on sperm concentration ranged from 128-343 x 10 6 sperm/ml in Large White and between 149-444 x 10 6 sperm / ml in Landrace boars; 3. percentage of mobile sperm in case of first batch ranged from 55-80% for Large White boars and between 55-87% for Landrace breed; 4. analyzing sperm viability values for both breeds of pigs in group I, shows that this parameter ranged from 65-90%; 5. in terms of morphological examination, normal sperm were found in the proportion of 85-95% for the two breeds of boars included in the experiments in group I; 6. in group I, primary abnormalities ranged from 3 to 4.2% in Large White and between 2.6 to 4% in Landrace breed, the secondary abnormalities between 7.6-8% in Large White and between 5-7.4% in Landrace boars; 7. regarding sperm head length there is a slight difference in favor of Landrace boars (6.96 to 8.75 µm) compare with Large White boars (6.50 to 8.60 µm); 8. sperm head width ranged from 2.80 to 3.94 µm in Large White boars and between 3 to 4.10 µm in Landrace boars, 9. in group II, the volume of collected semen ranged from 63-230 ml in Large White boars and Landrace boars between 74-250 ml; 10. for group II, sperm concentration varied in large limits, often exceeding the value of 300 x 106 sperm / ml; 11. motility before conservation was between 70-95% in Large White and between 75-95% in Landrace breed; 12. sperm viability ranged from 74-95% for Large White boars and between 78-95% for the Landrace breed; 13. the average percentage of morphologically normal sperm detected with Spermac method ranged from 89.8 to 94% for both breeds of pigs included in the experiment; 14. sperm abnormalities were apparent at Spermac stain, which facilitated the identification of both the primary secondary abnormalities; 15. from the study citomorfometric sperm head were found minor differences between length and width, average for the parameters studied fits within limits considered normal for swine species; 16. evaluation of freezing media based on BTS allowed the maintenance of sperm mobility between 25-26 % after freezing; 17. sperm viability ranged from 28 to 31%; 18. percentage of normal sperm morphology was recorded between 86 to 91.5%; 19. evaluation evaluation of freezing media based on MERCKIII allowed to obtain a sperm mobility between 25 to 28.1%; 20. sperm viability ranged from 30-34% ; X

21. freezing boar semen allowed to obtain good percentage of normal sperm between 89-93%; 22. freezing media, based on ANDROHEP Plus has allowed maintaining the mobility of sperm from 26.6 to 28.1%; 23. sperm viability in ANDROHEP Plus mediums ranged from 31-34%; 24. freezing boar semen allowed to obtain good percentage of normal sperm between 89-92%; 25. morphocytometric analysis revealed normal values of length and width of sperm head with discrete reduction trend after freezing; 26. the return percentage was positive in batches I and II (0) compared with groups (III and IV) subjected to estrus synchronization were the values recoded were between 6.66 and 8.88%; 27. installation of gestation in batches artificially inseminated with diluted semen was 91.11% in group I and 80% in group III; 28. at the batches artificially inseminated with frozen semen pregnancy was confirmed in 75.55 percent in group II and 68.88 in group IV; 29. when using frozen semen highest pregnancy rate was achieved with the A 2 medium (26.66%), followed by the other two where the values ranged between 22.22-24.44%; 30. the average number number of born piglets was higher in groups that artificial insemination was performed with diluted semen (group I 11.98 piglets/sow, group III - 11.15 piglets/sow) compared with groups who used semen frozen (group II 9.92 piglets/sow, group IV 9.70 piglets/sow); 31. regarding the number of weaned piglets, the values recorded in groups I and III were between 9.65 and 10 weaned piglets /sow and in batches II and IV between 8.15 and 8.51 weaned piglets /sow. PRACTICAL RECOMMENDATIONS 1. Due to the extraordinary scientific performance of biotechnology in veterinary medicine field we consider that the various specific news bioengineering and biotechnology must be applied with more openness. We recommend the introduction of morphocytometric semen compartment in cytological semen analysis (part of andrology sheet), considering that the cytological spermogram must be done periodically. 2. We also recommend the use and interpretation of the usual and special spermograms, conformingly all parameters must be comparatively correlated and evaluated, without any exacerbation of only one parameter importance. XI

3. The results reported in the research indicates that the preservation of boar semen reduces over time, semen quality in terms of mobility, viability, morphology and morphocytometry. We therefore recommend performing freezing boar semen for only for experimental purposes. 4. The modest results obtained with the use of frozen semen allows us to recommend its use only when it is impossible to achieve artificial insemination of sows with fresh or diluted semen. SELECTED REFERNCES 1. BIANCHI, I., K. CALDERAM, E.F. MASCHIO, E.M. MADEIRA, R. DA ROSA ULGUIM, C.D. CORCINI, D.C. BONGALHARDO, E.K. CORREA, T. LUCIA JR., J.C. DESCHAMPS, M.N. CORREA, 2008, - Evaluation of amides and centrifugation temperature in boar semen cryopreservation. Theriogenology 69, 632 638, 2008; 2. BWANGA, C.O., M.M DE BRAGANCA, S. EINARSSON, H. RODRÍGUEZ-MARTÍNEZ, 1990, Cryopreservation of boar semen in miniand maxi- straws. J. Vet. Med. A., 37: 651-658. 3. CARBAJO, G., C. CUELLO, M. RUIZ, J.M. VÁZQUEZ, E.A. MARTÍNEZ, J. ROCA, 2004, Effects of centrifugation before freezing on boar sperm cryosurvival. J. Androl., 25: 389-396. 4. CRABO, B.G., S. EINARSSON, 1971, Fertility of deep frozen boar spermatozoa. Acta Vet. Scand., 12: 125-127. 5. CURRY, M.R., 2000, Cryopreservation of semen from domestic livestock. Reviews Reprod., 5: 46-52. XII

6. GRAHAM, E.F., A.H.J. RAJAMAN, M.K.L. SCHMEHL, M. MAKI- LAURILA, R.E. BOWER, 1971, Fertility studies with frozen boar spermatozoa. A. I. Diesgest., 19: 61. 7. GROZA, I., I. MORAR, 2004, Andrologie veterinară, Ed. Gryphon, Braşov; 8. JOHNSON, L.A., 1985, Fertility results using frozen boar spermatozoa: 1970 to 1985. En: Deep freezing of boar semen. Eds: Johnson, L.A. y Larsson, K. Swedish university of agricultural sciences, Uppsala, Suecia. Pp: 199-222. 9. JOHNSON, L.A., 1998, Current developments in swine semen: preservation, artificial insemination and sperm sexing. Proc. 15th Cong. Int. Pig. Vet. Soc. 1: 225-230. 10. JOHNSON, L.A., K.F. WEIZE, P. FISER, W.M.C. MAXWELL, 2000, Storage of boar semen. Anim. Reprod. Sci.,62: 143-172. 11. POLGE, C., 1985, Sperm freezing, past, present and future. En: Deep freezing of boar semen. Eds: Johnson,L.A. y Larsson, K. Swedish university of agricultural sciences, Uppsala, Suecia. Pp: 167-173. 12. PURSEL, V.G., L.A. JOHNSON, 1971, Procedure for the preservation of boar spermatozoa by freezing. USDAARS bull, 44: 1-5. 13. ROCA, J., M. HERNANDEZ, G. CARVAJAL, J.M. VAZQUEZ, E.A MARTINEZ, 2006, Factors Influencing Boar Sperm Cryosurvival. Journal Animal Sciens 84: 2692-2699. 14. SARAVIA, F., M. HERNÁNDEZ, M. WALLGREN, A. JOHANNISSON, H. RODRÍGUEZ-MARTÍNEZ, 2007, Controlled cooling during semen cryopreservation does not induce capacitation of spermatozoa from two portions of the boar ejaculates. Int. J. Androl., 30: 485-499. 15. SILVA, P.F.N., B.M. GADELLA, 2006, Detection of damage in mammalian sperm cells. Theriogenology, 65:958-978. 16. THURSTON, L.M., W.V. HOLT, P.F. WATSON, 2003, Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison. Theriogenology, 60: 101-113. 17. VYT, P., D. MAES, E. DEJONCKHEERE, F. CASTRYCK, A. VAN SOOM, 2004, Comparative study on five different commercial extenders for boar semen. Reprod. Dom. Anim., 39: 8-12. 18. WOELDERS, H., 2005, Fundamentals and recent development in cryopreservation of bull and boar semen. Vet.Quart., 19: 135-138. XIII