The effect of enzymes on semen viscosity in Llamas and Alpacas

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1 Small Ruminant Research 38 (2000) 91±95 Technical note The effect of enzymes on semen viscosity in Llamas and Alpacas P.W. Bravo a,*, M. Ccallo b, J. Garnica b a Centro Experimental La Raya, Universidad Nacional San Antonio Abad, Cusco, Peru b Facultad del Medicina Veterinaria y Zootecnia, Universidad Nacional del Altiplano, Puno, Peru Received 1 October 1999; accepted 10 March 2000 Abstract The effect of four enzymes: collagenase, brinolysin, hyalurodinase, and trypsin were recorded on the viscosity, motility, percent live spermatozoa and acrosome integrity of Llama and Alpaca semen. Semen samples were collected using a modi ed arti cial vagina for each of the ve llamas and ve alpacas. A 25% solution of the of enzyme at a concentration of 1 mg/ml was added to the ejaculate. Analysis of variance was used to determine differences in eliminating viscosity and alterations in motility, percent live spermatozoa and the acrosomal integrity at 0 (time of semen collection), 2 and 5 min. In Llama and Alpaca semen, collagenase eliminated viscosity in 100 and 99% of the samples, respectively. Correspondingly, brinolysin in 89 and 59%; hyalurodinase in 88 and 36%; and trypsin in 55 and 68% of the samples (p<0.05). In the Llama sperm, motility decreased (p<0.05) with the addition of brinolysin (28%), trypsin (13%), hyalurodinase (12%), and collagenase (4%). In Alpaca semen, the enzymes used had no effect on sperm motility. Percent live spermatozoa variably decreased after the addition of brinolysin, hyalurodinase and trypsin. There was no signi cant difference in the acrosome integrity in Llama and Alpaca makes following the addition of the enzymes. Overall, collagenase had little or no in uence in decreasing motility, percent live spermatozoa and acrosome integrity, whereas, it was effective in eliminating semen viscosity. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Viscosity; Semen; Motility; Alpaca; Llama 1. Introduction Llama and Alpaca semen are extremely viscous after exposure to air, spermatozoa are embedded within a gelatinous material (San-Martin et al., 1968; Garnica et al., 1993), and it is dif cult to assess semen characteristics by making a smear on a glass slide. Semen of other species which form a coagulum similar to that of Llamas and Alpacas contain enzymes that intervene in the process of degeli cation. * Corresponding author. Present address: Dept. Anim. Sci., Brigham Young University, Provo, UT84602, USA. Tel.: address: pwbravo@hotmail.com (P.W. Bravo) Amongst the enzymes, a collagenase-like peptidase has been isolated in human, rat and bull semen (Koren and Milkovic, 1973). Fibrinolysin is found in human semen (Huggins and Neal, 1942), trypsin in semen from guinea-pigs and rabbits (Freund, 1958; Stambaugh and Buckley, 1970). The use of proteolytic enzymes, trypsin and collagenase, has been useful in the process of degeli cation in monkeys (Hoskins and Patterson, 1967), guinea-pigs (Freund, 1958), and more recently in Alpacas (Bravo et al., 1999). Nevertheless, it is important to study the precise time required for the elimination of viscosity as well as the effects on motility, percent live spermatozoa and acrosome integrity in the Llama and Alpaca. The main goal of the present study was to determine the time of /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (00)

2 92 P.W. Bravo et al. / Small Ruminant Research 38 (2000) 91±95 viscosity elimination (2 and 5 min), and the effect of collagenase, brinolysin, hyalurodinase and trypsin on motility, percent live spermatozoa and acrosome integrity of Llama and Alpaca semen. 2. Materials and methods Ten adult intact males ( ve Llamas and ve Alpacas) were used in this study. Animals were maintained on natural pastures at the La Raya research station, which is located at 4200 m above sea level, and 158S latitude and 708W longitude. Males were trained to copulate with a dummy female in which a modi ed arti cial vagina was placed using procedures developed by Bravo et al. (1997). Brie y, Alpaca dummies in a copulatory position (sternal recumbency), were placed where males were mating live females. After one normal mating, males were directed to copulate with a dummy. The arti cial vagina consisting of a 25 cm PVC pipe with a straight latex liner with a 15 ml graduated tube connected to the opposite end was used to collect semen from bucks. The whole arti cial vagina was wrapped in an electric blanket to maintain it at 378C for the period of the copulation (approximately 25 min). Semen was collected and then assessed for motility. Motility was assessed as the percentage of spermatozoa showing oscillatory movements of the tail. Four enzymes, collagenase (EC ), brinolysin (EC ), hyalurodinase (EC ), and trypsin 2.5% (EC ) from SIGMA Laboratories (Sigma Chemical Co., St. Louis, MO, USA) were diluted to a concentration of 1 mg/ml with distilled water and maintained at 48C. Enzyme solutions were mixed in a proportion of 25% of the ejaculate volume. Viscosity, motility, percent live spermatozoa and acrosome integrity were recorded at the time of semen collection (Time 0), 2 and 5 min after addition of the enzyme. Viscosity was measured indirectly and in the following manner: 50 ml of a semen sample was pipetted using a micropipette, 25 m1 was placed on a glass slide, and then pulled upward to compare the length of the thread on a ruler. The thread was pulled slowly until it broke. This distance was recorded as the viscosity of the semen sample. This operation was repeated at 2 and 5 min. Percent live spermatozoa were determined by staining semen samples with the Hancocks stain (Society of Theriogenology, Hastings, NE). Acrosome integrity was determined using a phase contrast microscope by examining the semen samples stained with modi ed Giemsa. Intact acrosomes were de ned as an uninterrupted membrane on two thirds of the spermatozoa head. Damaged acrosomes were de ned as an interrupted or broken membrane. A random sample of 100 spermatozoa was used to determine motility, live spermatozoa and acrosome integrity. A linear model that included the effects of enzymes, time and interactions were used to calculate differences. Data was analyzed using a computer software (NCSS, Kaysville, UT). When signi cant differences were detected means were compared using the Tukey test. 3. Results All fresh collected semen samples were viscous. The mean distance of the thread viscid semen was 1.5 cm with a range of 0.4±4.9 cm. Collagenase was more effective than brinolysin, hyalurodinase and trypsin in eliminating Llama and Alpaca semen viscosity (Fig. 1, Panels A and B). Once semen samples were in contact with the enzyme, viscosity diminished and the effect was irreversible. The mean motility in Alpaca semen samples was 68.2% and in Llama 66.6%. Collagenase did not signi cantly in uence semen motility. In contrast, brinolysin, hyalurodinase and trypsin (Fig. 1, Panels C and D) had a signi cant (p<0.05) effect. There was no change in individual motility. Fresh spermatozoa moved in place with no progressive motion. Llama and Alpaca spermatozoa did not show progressive movement even after 5 min of enzyme addition. The effect on percent live spermatozoa was less (p<0.05) in semen treated with collagenase (7.0%) than brinolysin (15.1%), hyalurodinase (15.2%) and trypsin (16.3%; Fig. 1, Panels E and F). The mean live spermatozoa was 74.9% for Alpaca and 73.3% for Llama semen. The effect on acrosome integrity was similar in semen treated with collagenase (6.1%) than brinolysin (8.4%), hyalurodinase (6.8%) and trypsin (6.3%; Fig. 1, panels G and H) and acrosomes appeared damaged in the anterior part.

3 P.W. Bravo et al. / Small Ruminant Research 38 (2000) 91±95 93 Fig. 1. Effect of collagenase (*), brinolysin (*), hyalurodinase (!) and trypsin (5) on the viscosity (A and B), motility (C and D), live spermatozoa (E and F), acrosome integrity (G and H) of Llamas (A, C, E and G) and Alpacas (B, D, F, H) semen. 4. Discussion This paper demonstrates the effect of some hydrolytic enzymes on viscosity, sperm motility, percent live spermatozoa and acrosome integrity in Alpaca and Llama semen samples. Since, semen from Llama and Alpaca are highly viscous, routine evaluation is seriously delayed and hampered. Semen viscosity was eliminated rapidly and in an irreversible manner following the addition of collagenase, brinolysin,

4 94 P.W. Bravo et al. / Small Ruminant Research 38 (2000) 91±95 hyalurodinase and trypsin. The enzymes were more effective on the Alpaca semen samples than in the Llama samples. This may be due to a greater viscosity of Llama semen compared to the Alpaca semen. Although, effect on viscosity was rapid, the effect was time dependant. Collagenase acted faster than the other enzymes used in this study. Although diverse methods of viscosity elimination have been reported, the best method found, was incubating semen samples in a water bath at 378C (Leyva et al., 1984) for at least 8 h in Alpaca (Garnica et al., 1993) and 22 h in Llama semen (Fuentes, 1990). The method of adding enzymes as reported in the present study, appears to be the most satisfactory thus far. The results of this study are in agreement with elimination of the coagulum in semen of monkeys (Hoskins and Patterson, 1967), guinea-pigs (Freund, 1958), Alpacas (Bravo et al., 1997), and Llamas (Bravo et al., 1996). Motility was affected minimally by the different enzymes used in this study. There was no change on the individual motility. Movement of spermatozoa continued to be in place before and after the addition of enzymes, i.e. no progressive motility. Even though semen samples were not viscous after enzyme addition, there was no signi cant change in motility. The attainment of progressive motility may be triggered by other mechanisms yet unknown. Nonetheless, a decrease in motility was not observed and therefore enzymes did not affect the normal motility of llama and alpaca spermatozoa. Similar observations were reported previously (Bravo et al., 1996), for monkeys (Roussel and Austin, 1967; Greer et al., 1968), and guinea-pigs (Freund, 1969). Percentage live spermatozoa were minimally affected by the addition of enzymes. The enzyme with the least effect on live spermatozoa was collagenase compared to brinolysin, hyalurodinase and trypsin. Similar results were reported previously in Alpacas (Bravo et al., 1998), Llamas (Bravo et al., 1996), and monkeys (Hoskins and Patterson, 1967; Srivastava et al., 1981), and guinea-pigs (Freund, 1969). The effect on the acrosome was not related to the enzyme used, neither to the time used in this study. Most of the acrosome damage was in the anterior part. The results found in this study are in agreement with previous results in bulls, rabbits (Bamba and Gran, 1988), rams, boars (Tamuli and Watson, 1994), and Alpacas (Bravo et al., 1999). 5. Conclusion The enzymes used in this study were effective in eliminating semen viscosity. Collagenase produced less effect on motility, live spermatozoa and acrosome integrity in llamas and alpacas, and appears the enzyme to be used in the process of semen evaluation. References Bamba, K., Gran, D., Effect of rapid warming of bull and rabbit semen. J. Reprod. Fertil. 82, 501±507. Bravo, P.W., Enriquez, E., OrdonÄez, C., El efecto de la tripsina y tres dilutores en semen de alpacas (The effect of trypsin and three extenders in alpaca semen). Allpak'a 6, l9±22. Bravo, P.W., OrdonÄez, C., Alarcon, V., Processing and freezing of semen of alpacas and llamas. Proc. 13th ICAR, Sydney, Australia 2, pp. 2±3. Bravo, P.W., Pacheco, C., Vilcapaza, L., Quispe, G., OrdonÄez, C., Degeli cation of alpaca semen and effect of dilution rates on arti cial insemination outcome. Arch. Androl. 43, 239±246. Freund, M., Collection and liquefaction of guinea-pig semen. Proc. Soc. Exp. Biol. Med. 98, 538±540. Freund, M., Interrelationships among the characteristics of guinea-pig semen collected by electroejaculation. J. Reprod. Fertil. 19, 393±403. Fuentes, C., Concentracion de los principales componentes quimicos del plasma seminal de la llama (Main chemical components of seminal plasma on llamas). M.V.Z. Thesis, Universidad Nacional del Altiplano, Puno, Peru. Garnica, J., Achata, R., Bravo, P.W., Physical and biochemical characteristics of alpaca semen. Anim. Reprod. Sci. 32, 85±90. Greer, W.E., Roussel, J.D., Austin, C.R., Prevention of coagulation in monkey semen by surgery. J. Reprod. Fertil. 15, 153±155. Hoskins, D., Patterson, D., Prevention of coagulum formation and recovery of motile spermatozoa from Rhesus monkey semen. J. Reprod. Fertil. 13, 337±340. Huggins, C., Neal, W., Coagulation and liquefaction of semen. J. Exp. Med. 76, 527±541. Koren, E., Milkovic, S., Collagenase-like peptidase in human, rat and bull spermatozoa. J. Reprod. Fertil. 32, 349± 356. Leyva, V., Sumar, J., Franco, E., Estudio preliminar de la concentracion de espermatozoides de semen de alpaca obtenido con vagina arti cial (Preliminary study of alpaca semen concentration obtained with arti cial vagina). 3ra. reunion anual de la Asociacion peruana de Produccion Animal, Lima. Roussel, J.D., Austin, C.R., Enzymic liquefaction of primate semen. Int. J. Fertil. 3, 288±290.

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