In vitro fertilizing capacity of frozen-thawed boar semen

ELSEVIER

IN VITRO FERTILIZING

CAPACITY OF FROZEN-THAWED

BOAR SEMEN

A. Cordova, ’ Y. Ducolomb,2 I. Jimenez,2 E. Casas2 E. Bonilla2 and M. Betancourt2a ‘Department of Agriculture and Animal Production, Metropolitan Autonomous University-Xochimilco, Mexico, D.F. 2Department of Health Sciences, Metropolitan Autonomous University-Iztapalapa, Mexico, D.F. Received for publication: Accepted:

February September

22 9 1996 23,

1996

ABSTRACT We describe a porcine semen cryopreservation technique and assess the in vitro fertilizing capacity of the frozen-thawed spermatozoa. The thawed spermatozoa did not lose the physiological properties of motility, viability, and acrosome reaction or capacity to fertilize in vitro. Immediately after thawing, the spermatozoa showed 5 1% mean motility, 60% viability, and 5% induced acrosome reaction. After 2.5 h of incubation in TALP medium, the spermatozoa exhibited 61% motility, 63% viability and 40% induced acrosome reaction, The average in vitro fertilization capacity of thawed spermatozoa was 68% compared with that of spermatozoa from fresh semen (85%). The percentage of polyspermy was highly variable, with frozen-thawed samples ranging from 0 to 28% and fresh samples from 0 to 30%. The results obtained with frozen semen from 5 boars of different breeds did not show considerable variation. This suggests that the freezing-thawing technique is reproducible and adequate for in vitro fertilization, 0 1937 by Elsevier Science Inc. Key words: semen, cryopreservation, in vitro fertilization, boar INTRODUCTION Freezing and thawing of gametes was first successfully developed in 1949 when Polge et al. (19) reported the cryoprotective property of glycerol. These authors successfully recovered spermatozoa from several mammalian species after freezing semen in a glycerol solution. Several techniques have been developed using glycerol or other cryoprotectants to preserve a great diversity of biological materials such as oocytes, spermatozoa, embryos and different somatic cells. These materials can be frozen for several months without significantly altering their original physiological characteristics (1). The use of stored gametes has promoted the propagation of the

Acknowledgments We wish to thank Slaughterhouse ABC, Los Reyes, Mexico, for providing the porcine ovaries; and the Veterinary School of the National University of Mexico for providing the semen samples. This study was supported in part by CONACYT, Mexico, by grants 1505-M9207. a To whom reprint requests should be addressed: Departamento de Ciencias de la Salud, Universidad Autonoma Metropolitana-Iztapalapa, C.P. 09340, Mexico, D.F., Mexico Theriogenology 47:1309-1317, 1997 0 1997 by Eisevier Science Inc.

0093-691x/97/$17.00 PII SOO93-691X(97)00123-4

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best animals in reproduction centers, even after the donor has died. In vitro fertilization (IVF), which was first performed in rabbits by Chang in 1959 (9), has made important contributions to the understanding of mammalian reproductive phenomena, which in turn has helped to optimize the efficiency of this technique. It has been used in several experimental animal models such as rats, hamsters, cats and dogs (l), and farm animals such as pigs, sheep and cows (1,17). In vitro fertilization has also been used in humans for assisted reproduction purposes (16). In vitro fertilization has been used in pigs for basic research, to understand the biology of mammalian gametes, from their maturation and interaction to the first stages of embryo development ($22). The technique can also be used in commercial breeding to reduce generation intervals and to produce genetically improved pigs (10). Combined with oocy-te and sperm freezing processes, IVF has helped to establish gene banks with commercially relevant genetic material (10, 17). Readily available cryopreserved semen would facilitate IVF. Frozen pig semen has been used mainly for artificial insemination, and only a few studies have described the use of cryopreserved pig semen for IVF, with contradictory results. Nagai et al. (17) unsuccessfully used cryopreserved ejaculated semen for IVF, whereas Zheng et al. (23) obtained better IVF results using cryopreserved ejaculated semen than fresh ejaculated semen. The purpose of the present study was to determine the in vitro fertilizing capacity of frozen-thawed porcine spermatozoa. MATERIALS AND METHODS All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless stated otherwise. Collection and Transportation of Semen Semen was obtained from 9 pigs (Duroc, Hampshire, Landrace, Spotted and DurocHampshire) of known reproductive history, with mean age 2.5 yr, from the Zapotitlan Porcine Experimental Farm of the School of Veterinary Medicine of the National University of Mexico. The ejaculate was obtained by the gloved hand method, and the gelatinous portion was separated by filtration through gauze. The volume, motility and concentration of spermatozoa were immediately assessed (12). Sample were transported at 37’C to the laboratory in less than 1 h after collection. Semen Freezing A sample volume containing 1.4 x109 spermatozoa was centrifuged at 350 g for 10 min to eliminate the seminal plasma (13). A cellular pellet was diluted to 4.5 ml with diluent A (200 nib4 Tris, 70.8 mM citric acid, 55.5 1 mM glucose, 25% egg yolk and 0.1% dihydrostreptomycin) at room temperature. The sperm suspension was kept at 5°C for 1.5 h (8). Afterwards, 2.25 ml of

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diluent B at 5°C were added (200 mM Tris, 70.8 mM citric acid, 55.5 1 mM glucose and glycerol at a final concentration of 5%) and kept at Y’C for 15 min. An equal volume of diluent B at 5°C was then added, and after 3 h of equilibration at 5°C (8) the samples were placed in 0.25ml straws and sealed with polyvinyl alcohol. The straws containing a final concentration of 160 x lo6 spermatozoa/ml were placed in liquid nitrogen vapors approximately 3 cm from the liquid for 9 min, then plunged in liquid nitrogen and stored until used. The storage time ranged from 2 wk to 1 yr Thawing and In Vitro Capacitation The straws were thawed in a 5O’C water bath for 20 set and emptied into Tyrode’s medium supplemented with 0.25 mM sodium pyruvate, 6 mg/ml BSA, 21.6 mM sodium lactate, and 10 mM HBPES (TALP-HEPES; 2) at 37°C to a final volume of 1.5 ml. Spermatozoa were allowed to swim up to this medium during a 30-min incubation at 37°C to select the motile and mainly acrosome-intact fraction of the sperm population (18). The migrated spermatozoa were then recovered from the tubes with the upper 0.5 ml of medium. A further dilution of the motile fraction of spermatozoa was made in TALP-HEPES, to a final concentration of 1 x lo7 spermatozoa/ml. Aliquots of 0.5ml were incubated in wells of a 4-well plate (Nunc, Roskilde, Denmark) at 37°C in a humidified 5 % CO2 atmosphere for 2.5 h, conditions which support in vitro pig sperm capacitation (6). Analysis of Sperm Viability Sperm viability was assessed in a sample of 100 spermatozoa by supravital staining with the Hoechst 33258 dye (bis-benzimide; Sigma). Suspensions of washed spermatozoa were incubated for 8 min at 37°C with Hoechst diluted in PBS at a concentration of 1 l&ml. The free dye was washed from the sperm suspension by 2 cycles of centrifirgation (500 g for 10 min), and the pellet was resuspended in fresh PBS. The spermatozoa were then used immediately for smear preparations (14). Analysis of Sperm Motility Sperm motility was assessed at x 400 with an inverted microscope by counting the percentage of progressively motile spermatozoa in a sample of 100 sperm cells (3). Assessment of the Acrosome Reaction The number of acrosomally responsive spermatozoa was determined by exposing the sperm cells to solubilized pig zona pellucida immediately after thawing, and after 2.5 h of incubation in TALP-HEPES medium at 37’C. The solubilized pig zona pellucida was prepared from oocytes isolated from ovaries by using nylon screens as previously described (11). Spermatozoa were incubated in 1 ug Hoechst 33258/ml for 8 min at 37°C centrifuged in 2% polyvinyl pyrrolidone (MWav 40 000) in PBS to remove the stain, and fixed in 4°C ethanol for 30 min. Slides were prepared and air-dried. Spermatozoa were stained with 100 ~1 fluorescein

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isothiocyanate conjugated Pisum sativum agglutinin (FITC-PSA, 200 ug/ml in PBS) at 37°C for 20 min, washed with distilled water, and analyzed on a Zeiss epifluorescence microscope at x 1000 with a wide blue light excitation filter system for fluorescein (450-490nm), and an ultraviolet excitation filter system for the Hoechst stain (200 to 400 nm). At least 200 spermatozoa were counted per slide. Staining of pig spermatozoa with FITC-PSA result in 3 alternative fluorescence patterns: uniformly stained spermatozoa throughout the acrosomal region (acrosome-intact), spermatozoa remaining unstained (acrosome reacted), and spermatozoa showing a band of residual flluorescence corresponding to the equatorial segment (acrosome-reacted). Only live spermatozoa were counted -those that did not allow penetration of Hoechst dye and thus were not stained blue (4). In Vitro Fertilization Ovaries, obtained from swine at the ABC slaughterhouse in Los Reyes, Mexico, were transported in less than 2 h to the laboratory in 0.157 M NaCl at 37°C. Oocytes with cumulus cells were aspirated from 3- to 6- mm diameter follicles with a 21-gauge needle attached to a 3-ml syringe (Becton Dickinson, Mexico City, Mexico); 15 to 20 oocytes were incubated in a 60-ul drop of maturation medium: TC199 medium supplemented with 10% inactivated fetal calf serum (Microlab, Mexico City, Mexico), 1 mg/ml glucose (Baker, Mexico City, Mexico), 2 III/ml Pergonal (LH, FSH; Serono, Mexico City, Mexico), 0.25 mM sodium pyruvate, 10 ug/rnl gentamycin (Scheramex, MexicoCity, Mexico), and 1 ug/ml g-estradiol (280 to 290 mOsm/kg). The microdrops were placed in 4-well plates (Nunc) and covered with pa&in oil .Oocytes were incubated at 37’C in a humidified atmosphere with 5% CO2 for 48 h, then coincubated with 5 x lo4 spermatozoa per well in a TALP fertilization medium microdrop (5) for 24 h under the above conditions. The oocytes were fixed in 3:l methanol-acetic acid for 1 h, stained with 2% acetic orcein and examined under a bright field microscope to assess fertilization. Oocytes showing 2 pronuclei were considered to be fertilized. Statistical Analysis Sperm motility and viability and the induced acrosome reaction in fresh and frozen-thawed semen were compared by analysis of variance. The percentage of fertilized oocytes with fresh and frozen-thawed spermatozoa were compared with the Mann-Whitnney U-test, Statistical significance was set at P< 0.05. RESULTS Only fresh semen showing sperm motility greater than 60% before beginning the freezing process was used. Motility, viability, induced acrosome reaction and in vitro fertilization capacity were assessed after thawing. Table 1 shows the motility of thawed spermatozoa, from 9 boars immediately after thawing and atIer 2.5 h of incubation in TALP medium at 37’C. Mean motility was 5 1% in recently thawed spermatozoa, which was significantly lower than the 80% mean motility of fresh sperm samples. However, after 2.5 h of incubation in TALP medium, frozen-thawed spermatozoa showed an

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average of 61% motility, which was not significantly different than that of fresh spermatozoa (68%). Mean viability of frozen-thawed spermatozoa immediately after thawing and after 2.5 h of incubation in TALP medium was similar (60 and 63%, respectively); while it was significantly higher in fresh semen samples (82 and 74%, respectively; Table 2). The percentage of spermatozoa responding to solubilized pig zona pellucida at the beginning of capacitation was not significantly different between frozen-thawed semen (5%) and fresh semen (3%). However, the percentage of acrosome-reacted spermatozoa after the capacitation process was higher in frozen-thawed semen (40%) than in fresh semen (20%; Table 3). Table 4 shows IVF capacity of frozen-thawed and fresh spermatozoa observed in 6 different replicates. The average IVF capacity of fresh spermatozoa was 85%, which was significantly higher than that obtained with frozen-thawed spermatozoa (68%). In 6 assays, the percentage of polyspermy was highly variable, ranging from 0 to 28% in frozen-thawed sperm samples and 0 to 30% in fresh samples.

Table 1, Motility of frozen-thawed and fresh spermatozoa before and after capacitationa Frozen-thawed spermatozoa

Fresh spermatozoa

Replicateb

% Motility before capacitation

% Motility after capacitation

% Motility before capacitation

% Motility after capacitation

1 2 3 4 5 6 7 8 9 Mean + SD

50 40 50 50 50 60 50 50 60 51 +6

50 70 50 50 70 60 60 70 70 61 f 9.3

80 80 70 80 90 80 70 80 90 80 + 7.1

70 70 60 70 60 60 70 80 70 68 f 6.6

aFrozen-thawed sperm motility was assessed at x 400 with an inverted microscope immediately after thawing (before capacitation) and after 2.5 hours of incubation in TALP medium at 37°C (after capacitation). bEach replicate was carried out on different days using frozen-thawed spermatozoa from 9 different pigs.

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Table 2. Viability of frozen-thawed and fresh spermatozoa before and after capacitationa Frozen-thawed spermatozoa Replicateb

% Viability before capacitation

1 2 3 Mean f SD

57 67 56 60 f 6.1

% Viability after capacitation 67 64 59 63f4

Fresh spermatozoa % Viability before capacitation

% Viability after capacitation

84 77 85 82 + 4.4

69 78 75 74 k 4.6

aviability of frozen-thawed spermatozoa was assessed by staining with Hoechst 33258 immediately after thawing (before capacitation) and after 2.5 hours of incubation in TALP medium at 37°C (after capacitation). Only spermatozoa that did not stain blue were considered to be live. bEach replicate was performed on different days, using frozen-thawed spermatozoa from 3 different pigs.

Table 3. Zona pellucida-induced acrosome reaction in frozen-thawed and fresh spermatozoa before and after capacitationa Frozen-thawed spermatozoa Replicateb

% Acrosome reaction before capacitation

1 2 Mean k SD

4 6 5 * 1.4

% Acrosome reaction after capacitation 42 39 40+2.1

Fresh spermatozoa % Acrosome reaction before capacitation

% Acrosome reaction after capacitation

1 5 3 IfI2.8

17 23 20 * 4.2

aThe acrosome reaction was induced in frozen-thawed spermatozoa with solubilized pig zona pellucida, immediately after thawing (before capacitation) and after 2.5 hours of incubation in TALP medium at 37°C (after capacitation) and was assessed by fluorescein-conjugated &JRI sativum agglutinin staining. bEach replicate was performed on different days using frozen-thawed spermatozoa from 2 different pigs.

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Table 4. Oocytes fertilized in vitro with frozen-thawed and fresh spermatozoaa Frozen-thawed spermatozoa Replicateb

Number of oocytes fertilized using fiozenthawed semen (%)

% polyspermic oocytes

Fresh spermatozoa Number of oocytes fertilized using fresh semen

% polyspermic oocytes

(%) 1 2 3 4 5 6 Mean + SD

16 (94) 12 (57) 28 (80) 17 (57) 15 (65) 20 (56) 68* 14

6.25 0 28.6 23.5 0 15

16 (89) 8 (89) 11 (100) 10 (71) 6 (86) 14 (74) 85 i9

6.25 0 0 30 0 14.3

aoocytes showing at least 2 pronuclei were considered to be fertilized. bEach replicate was performed on different days using frozen-thawed semen from 6 different pigs.

DISCUSSION Studies on the functional characteristics of frozen-thawed pig spermatozoa have reported variable results. Several diluents, cryoprotectants and recipients have been tested to improve the freezing-thawing process. In the present study, we adapted several methods in order to obtain adequate functional characteristics of cryopreserved boar semen for the purpose of then using this semen in IVF. After capacitation in TALP medium, there was no difference in motility between cryopreserved and fresh spermatozoa. However, sperm viability as assessed by dye exclusion, was significantly lower in the cryopreserved spermatozoa (63 vs 74%). Sankai et al. (20) also observed that frozenthawed monkey spermatozoa have a shorter life span than fresh spermatozoa. Cryopreserved sperm cells show better response to the acrosome reaction inducer. After 2 h of capacitation, the percentage of acrosome-reacted cryopreserved spermatozoa was 40%, while that of fresh spermatozoa was 20%. These results are similar to those of Sankai et al. (20) who reported that freezing-thawing seems to reduce the time required for monkey sperm capacitation. The mean percentage of fertilized oocytes by frozen-thawed ejaculated spermatozoa was 68%, while it was 85% for fresh spermatozoa, as assessed by pronuclei formation. This differs from the report of Nagai al. (17) who did not observe penetration when using cryopreserved ejaculated spermatozoa. Theabove authors were successful only when they used cryopreserved epididymal spermatozoa, achieving 40% penetrated oocytes and 22% oocytes with pronuclei formation. On the other hand,Zheng et al. (23) successfully performed IVF using frozen-thawed ejaculated pig

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spermatozoa, obtaining 68% penetrated oocytes and a maximum of 45% oocytes with pronuclei formation. Using fresh semen they observed an 80% sperm penetration rates, but only 8% of the oocytes showed pronuclei formation. In the present study, we achieved high IVF efficiency with both fresh and frozen-thawed ejaculated semen. Using a final concentration of 8.3 x lo5 sperm/ml isolated by the swim-up method, we observed a low percentage of polyspermy with both cryopreserved (9%) and fresh semen (8%), similar to the findings by Nagai et al. (17). They observed 10% polyspermy using cryopreser-ved epididymal spermatozoa. However, Zheng et al. (23) reported a wide variation in polyspermy (8 to 49%) that was dependent on sperm concentration and the separation process used for IVF. Observed differences in motility, viability, acrosome reaction and IVF between fresh and frozen-thawed sperm cells could be due to altered permeability of the sperm membrane induced by the cryoprotectants, which would drastically alter the function of the cell (7, 15, 21). Nevertheless, the results shown here and those of Zheng et al. (23) indicate that frozen-thawed spermatozoa can be successfully used for IVF in pigs, This may facilitate the use of IVF to increase pig production. Our findings may also contribute to the study of the mammalian fertilization process, REFERENCES 1. Bavister B. Applications of IVF technology. In: Bavister B, Cummins J, Roldan E. (eds), Fertilization in Mammals. New York: Plenum Press, 1990; 33 l-334. 2. Bavister BD. Yanagimachi R. The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol. Reprod. 1977; 16: 228-237. 3. Berger T, Turner KO, Meizel S, Hedrick JL. Zona pellucida induced acrosome reaction in boar sperm. Biol Reprod 1985; 40:525-530. 4. Berger T. Pisum sativum agglutinin used as an acrosomal stain of porcine and caprine sperm. Theriogenology 1990; 33 :689-695. 5. Betancourt M, Fierro R, Ambriz D. In vitro fertilization of pig oocytes matured in vitro. Theriogenology 1993;40:1155-1160. 6. Bonilla E, Amador A, Betancourt M. In vitro capacitation of pig spermatozoa in a protein-free medium supplemented with histidine and cysteine. Med Sci Res 1994; 22: 725-726. 7. Buhr MM, Curtis EF; Kakuda NS. Composition and behavior of head membrane lipids of fresh and cryopreserved boar sperm. Cryobiology 1994; 3 1:224-238. 8. Bwanga CO. Cryopreservation of Boar Semen, Studies on Freezing, Packaging and Fertilizing Capacity. Ph. D. Thesis. Swedish University of Agricultural Sciences. Faculty of Veterinary Medicine. Uppsala, Sweden, 1990. 9. Chang MC. Fertilization of rabbit ova in vitro. Nature 1959; 184:466-467. 10. Hammitt DG, Martin PA. Fertility of frozen-thawed porcine semen following controlled-rate freezing in straws. Theriogenology 1989; 32:359-367. 11. Hedrick JL, Wardrip NJ. Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes. Anal Biochem 1986; 157: 63-70. 12. King GJ, Macpherson JW. A comparison of two methods for boar semen collection. J Anim Sci 1973; 36:563-565.

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13. Maxwell WMC, Salamon S. Fertility of frozen-thawed boar semen. Aust J Biol Sci 1979, 32:243-249. 14. Mendoza C, Carreras A, Moos J, Tesarik J. Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil 1992; 95: 755-763. 15. Molinia FC, Evans G, Maxwell WMC. Incorporation of penetration cryoprotectans in diluents -freezing ram spermatozoa. Theriogenology 1994; 42:849-858. 16. Morshedi M, Dehinger S, Veer LL, Hakan E, Acosta AA. Cryopreserved/thawed for in vitro fertilization: results from fertile donors and infertile patients. Fertil Steril 1990; 54: 1093 -1099. 17. Nagai T, Takahashi T, Masuda H, Shioyo Y, Kumayama M, Fukushima M, Iwasaki S, Hanada A. In vitro fertilization of pig oocytes by frozen boar spermatozoa. J Reprod Fertil 1988; 84:585-591. 18. Parrish JJ, Susko-Parrish JL, LeibfXed-Rutledge ML. Critser ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986; 25: 591-560 19. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949; 164:666 abstr. 20. Sankai T, Terao Y, Yanagimachi R, Cho, F, Yoshikawa Y. Cryopreservation of spermatozoa from cynomolgus monkeys (Macaca fascicularis). J Reprod Fertil 1994; 101:273-278. 2 1. Thomas CA, Garner DL. Post-thaw bovine spermatozoa1 quality estimated from fresh samples, J Androl 1994; 15:489-500. 22. Yoshida M, Ishizaki Y, Kawagishi H. Blastocyst formation by pig embryos resulting from in vitro fertilization of oocytes matured in vitro. J Reprod Fertil 1990; 88: l-8. 23. Zheng YS, Fiser P, Sirard MA The use of ejaculated boar semen after freezing in 2 or 6% glycerol for in vitro fertilization of porcine oocytes matured in vitro. Theriogenology 1992; 38:1065-1075.