Efeito da coleta fracionada e de diluidores de sêmen resfriado a 5° C sobre a longevidade de espermatozóides de jumentos

abmvz Arquivo Brasileiro de Medicina Veterinária e Zootecnia Arq. Bras. Med. Vet. Zootec. 0102-0935 1678-4162 Universidade Federal de Minas Gerais, Escola de Veterinária Belo Horizonte, MG, Brazil O objetivo deste trabalho foi avaliar a longevidade dos espermatozóides provenientes da fração rica e da fração completa de ejaculados de jumentos, preservados em dois diluidores. As duas amostras seminais foram diluídas nos dois diluidores (Kenney e Baken modificado) e preservadas a 5° C. Sêmens de cinco jumentos foram coletados com auxílio de vagina artificial de fundo aberto, uma vez por semana e por cinco semanas consecutivas. Os dois primeiros jatos do ejaculado (fração rica) foram coletados separadamente do restante do ejaculado. Amostras de ejaculado completo foram obtidas misturando proporcionalmente parte da fração rica e parte do restante do ejaculado. Amostras de sêmen foram, em seguida, misturadas 1:1 com cada diluidor e mantidas em temperatura ambiente até a avaliação da concentração. A diluição final teve por objetivo a obtenção de 50×10(6) espermatozóides por ml. O sêmen diluído foi resfriado até 5° C numa taxa de -0.6° C/min. A motilidade total e progressiva e o vigor foram avaliados no final da diluição, após resfriamentos e a cada 24 horas até a queda da motilidade total abaixo de 10%. A morfologia espermática foi avaliada por microscopia de contraste de fase em amostras coletadas após a diluição, e nos dias 3, 6 e 9 pós-coleta. As médias foram comparadas pelo teste t de Student. Não houve diferença na longevidade dos espermatozóides entre a fração seminal rica e a fração completa com ambos os diluidores testados. Observou-se que os espermatozóides, tanto da fração rica como da completa, foram melhor preservados no meio Baken modificado (base de gema de ovo) do que no meio Kenney (base de leite desnatado). Effect of split ejaculation and seminal extenders on longevity of donkey semen preserved at 5° C [Efeito da coleta fracionada e de diluidores de sêmen resfriado a 5 °C sobre a longevidade de espermatozóides de jumentos] S.L.V. Mello, M. Henry*, M.C. Souza, S.M.P. Oliveira Unidade de Pesquisa Eqüídea - UFMG / EPAMIG Caixa Postal 567 30123-970 - Belo Horizonte, MGRecebido para publicação em 14 de julho de 1999 *Autor para correspondência E-mail: [e-mail] The aim of this study was to evaluate the longevity of donkey sperm comparing the rich seminal fraction and the whole semen in two extenders, Kenney and modified Baken extenders. Semen of five donkeys were collected through an open-end artificial vagina once a week for five consecutive weeks. The two first jets (rich fraction) of semen were collected separately from the rest of the ejaculate. Whole semen samples were obtained mixing proportionally part of the rich with part of the poor seminal fractions. Seminal samples were immediately diluted 1:1 in each extender and maintained at room temperature during sperm concentration analysis. Samples were further diluted to rich 50´106 sperm per ml, cooled in a refrigerator at the initial rate of –0.6° C/min and preserved at 5° C. Total motility (TM), progressive motility (PM) and sperm vigor (V) were examined after final dilution and cooling, and every 24 hours up to the decrease of total motility under 10%. Sperm morphology was evaluated using a phase contrast microscope directly after dilution, on days 3, 6 and 9 post collection. It was used a 2´2 factorial design in a randomised bloc experiment, and means were compared by Student’s t test. Longevity did not vary between the rich seminal fraction and the whole semen for both extenders used. TM, PM, V and sperm morphology were better preserved in the extender with egg yolk (modified Baken extender) than in the one with skimmed milk (Kenney) in both seminal fractions. Key words: Donkey, cooled semen, sperm longevity RESUMO O objetivo deste trabalho foi avaliar a longevidade dos espermatozóides provenientes da fração rica e da fração completa de ejaculados de jumentos, preservados em dois diluidores. As duas amostras seminais foram diluídas nos dois diluidores (Kenney e Baken modificado) e preservadas a 5° C. Sêmens de cinco jumentos foram coletados com auxílio de vagina artificial de fundo aberto, uma vez por semana e por cinco semanas consecutivas. Os dois primeiros jatos do ejaculado (fração rica) foram coletados separadamente do restante do ejaculado. Amostras de ejaculado completo foram obtidas misturando proporcionalmente parte da fração rica e parte do restante do ejaculado. Amostras de sêmen foram, em seguida, misturadas 1:1 com cada diluidor e mantidas em temperatura ambiente até a avaliação da concentração. A diluição final teve por objetivo a obtenção de 50´106 espermatozóides por ml. O sêmen diluído foi resfriado até 5° C numa taxa de –0.6° C/min. A motilidade total e progressiva e o vigor foram avaliados no final da diluição, após resfriamentos e a cada 24 horas até a queda da motilidade total abaixo de 10%. A morfologia espermática foi avaliada por microscopia de contraste de fase em amostras coletadas após a diluição, e nos dias 3, 6 e 9 pós-coleta. As médias foram comparadas pelo teste t de Student. Não houve diferença na longevidade dos espermatozóides entre a fração seminal rica e a fração completa com ambos os diluidores testados. Observou-se que os espermatozóides, tanto da fração rica como da completa, foram melhor preservados no meio Baken modificado (base de gema de ovo) do que no meio Kenney (base de leite desnatado) Palavras-chave: Jumento, sêmen resfriado, longevidade espermática INTRODUCTION The effect of seminal plasma on the sperm cells and on fertility has been studied in several species, however, no consensus has been riched (Mann, 1969; Magistrini et al., 1987; Martinus et al., 1991; Tischner & Kosiniak, 1992). In horses, the great majority of the authors agree that the maintenance of the whole seminal plasma fraction may be deleterious to in vitro sperm preservation (Pickett et al., 1975; Heiskanen et al., 1987; Jasko et al., 1991; Aurich et al., 1996). Since in this species fertilization may be riched with sperm from the cauda (tail) of the epydidimus, it is still not clear which is the real physiological significance of the seminal plasma (Garner & Hafez, 1993). In order to overcome the effect of seminal plasma, cooling and sperm toxic metabolic residues on in vitro sperm longevity, it is necessary to add semen extenders which provide nutrients and protection (Carvalho, 1994). A large variety of extenders has been developed to protect cooled and frozen equine semen (Palmer, 1984; Pickett & Amann, 1987; Varner et al., 1993; Silva Filho, 1994; Webb & Arns, 1995), however, the macromolecules found in the milk and egg yolk are still the most used nowadays. This study is aimed to evaluate the effect of decreasing seminal plasma fraction and of two seminal extenders, one based on milk and the other on egg yolk, on the longevity of donkey semen preserved at 5° C. MATERIALS AND METHODS The experiment was carried out during July and August. Five donkeys of the Nordestino and Pêga breeds ranging from three to eight year-old were used. Previous to the start of the experiment all donkeys were submitted to daily seminal collection for eight consecutive days in order to stabilize sperm extra gonadal reserve. During the experiment, each donkey was sampled once a week for five consecutive weeks. Open-end artificial vagina (Tischner, 1989) was used to split the rich fraction (first two jets) of the rest of the semen including the gel fraction. Both fractions were kept at 37ºC and immediate analyses of the total (TM) and progressive (PM) motility and vigor were performed. Each fraction (rich and whole semen) was then diluted 1:1 with two seminal extenders within 15 minutes of the semen collection. Whole semen samples were obtained mixing proportionally part of the rich with part of the poor seminal fraction. Concentration was estimated through hemocitometer and final dilution was aimed to obtain 50´106 sperm cells/ml. Skimmed milk (Kenney et al., 1975) and modified Baken extenders with no antibiotics were used. Baken extender was modified by adding 10% egg yolk instead of 3% as reported by Nishikawa (1959). Osmolality of the skimmed milk and egg yolk extenders were 352.5 and 357.0 mOsm/kg, respectively. A domestic fridge was used to cool seminal samples at a rate of –0.6° C/min and temperature of maintenance was 5° C. TM, PM and vigor (scale 1 to 5) were estimated under light microscope after final dilution and cooling and every 24 hours until TM was £10%. Sperm morphology was evaluated by phase contrast microscope in fresh seminal samples and in samples collected on days three, six and nine post-cooling. A 2´2 (2 extenders and 2 semen fractions) factorial design was used, and included five donkeys and the means of five ejaculates per donkey. Means and standard deviations were calculated and analysis of variance was carried out (Snedecor & Cochran, 1980). Means were compared by Student’s t test. Variables expressed in percentage (TM, PM) were transformed to the arc-sine X/100 angle prior analysis of variance. Analysis was performed for each observation time. RESULTS Mean values of TM, PM and vigor at each observation time and for each treatment are shown in Table 1. TM and PM of the rich seminal fraction and of total semen diluted with Kenney extender at the post-cooling observation time were lower (P<0.05) than those obtained in the rich fraction semen diluted in modified Baken extender. From the post cooling observation time on TM, PM and vigor showed to be statistically higher most of the time in modified Baken extender independently of the seminal fraction used. Total motility above 10% was observed for 168 hours for all treatments. There was no effect of the seminal fraction on longevity of the semen as defined herein (P>0.05). However, the modified Baken extender preserved motility better from 24 hours post seminal collection on. Yet, better vigor was observed in modified Baken extender from the post-cooling observation time on (P<0.05). On day seven post-seminal collection, only the total seminal fraction diluted in modified Baken extender still showed superior TM, PM and vigor than seminal fraction diluted in Kenney extender. There was no significant difference in incidence of abnormal sperm between the rich seminal fraction and the whole reconstituted semen. Total normal sperm, acrosome defect, head defect, middle piece defect, defect of the cauda (tail) and total protoplasmatic dropplet of the rich fraction were, respectively, 71.47± 18.88%, 0.36± 0.16%, 3.05± 0.3%, 13.2± 0.63%, 2.8± 0.54% and 9.12± 0.61%. Sperm morphology post-final dilution, on days three, six and nine is shown in Table 2. There was no influence of the extenders within seminal fraction on sperm morphology directly after final dilution (Table 2). On day three, independently of the seminal fractions, total number of abnormal sperm was statisticaly higher in semen preserved in Kenney extender. Increased abnormal acrosome and middle piece defects were the defects which contributed most to the changes in total abnormalities between the two extenders. A gradual increase (6 to 9%) of abnormal sperm was seen between day three to six post-collection in both seminal extenders, however, total abnormal sperm in semen preserved in modified Baken extender on day six was still lower than that found in sperm preserved in Kenney extender on day three. As there was no sperm alive on day nine in Kenney extender, no analysis of sperm morphology was performed. An increase in acrosome abnormalities in sperm preserved in modified Baken extender was the main change in the semen on day nine. There was no influence of seminal fractions on occurrence of sperm abnormalities at any time observed. Total and progressive motility and sperm morphology (abnormal) during observation time are shown in Figure 1. DISCUSSION Donkey ejaculates are characterized by a volume which varies between 25 to 45ml, most of the time with the sperm cells showing high total and progressive motility. Gel may be present in some ejaculates (Henry et al., 1987), but, apparently in lower proportion than that found in horses. In horses, the total volume of semen per ejaculate is larger and may vary between 40 to 150 ml (Pickett et al., 1976). In this study, in both extenders tested, sperm longevity and maintenance of sperm morphology were similar between diluted rich and diluted whole seminal fractions. The lack of finding a beneficial effect of retrieving most of the seminal fluid in sperm longevity in donkeys, could be explained by two hypotheses. The first one would be that the seminal plasma has no deleterious effect on in vitro sperm survival in donkeys. This hypothesis is in disagreement to what is commonly known for horses. It has been speculated that in the latter species that, for in vitro preservation, high proportion of seminal fluid on diluted semen is detrimental for maintenance of sperm viability (Pickett et al., 1975; Shannon & Curson, 1983; Jasko et al., 1991). The second hypothesis is related to the characteristics of donkey semen associated to the sperm dilution rate used in this study. As described above, ejaculates of donkeys, on the average, have a low volume and high sperm concentration. Due to these characteristics, the dilution rate used in this study, in order to obtain 50´106 sperm cells per ml, as stipulated in the protocol, may have diluted the seminal plasma above a threshold that could affect in vitro sperm survival. Independently of this reason, it was evident that fractionating the ejaculate did not bring any advantage in preserving donkey semen in vitro at 5° C. The major difference found between treatments was due to an effect of extender. Independently of fractionating or not the ejaculate, it was found that semen was better preserved in the modified Baken extender than in the Kenney extender. This difference, observed through the sperm motility and the morphology of the sperm cell, was already detected on day one post collection and persisted during the whole preservation time. A considerable decrease in total and progressive motility was already observed between day one and two in the Kenney extender, although, longevity, as herein defined, lasted up to day six to seven. In the modified Baken extender, the decrease in total and progressive motility occurred gradually up to day nine post collection, when motility was near but still above 10%. In the latter extender, by day seven to eight motility was still around 30%. The main change in sperm morphology during sperm preservation occurred at the acrosome and middle piece level. In both extenders, the alterations found seemed to be related to changes in the integrity of the sperm membrane. As far as it could be observed through the phase contrast microscope, the changes found at the acrosome level were more like those observed during the acrosome reaction, i.e., vesiculation at the cranial pole of the sperm head and rupture of the cell membrane. At the middle piece level, changes were more linked to an increase of the thickness of the region. In Figure 1, it seems that the increase in the incidence of abnormal sperm occurred as a decrease in sperm motility was detected. As sperm morphology was not evaluated every day, it was not possible to state which change occurred first. For both variables, motility and sperm morphology, the modified Baken extender showed to have a more efficient protective effect than the Kenney extender. By day three no change in the incidence of sperm abnormalities was found in the modified Baken extender, while by day nine, the incidence of sperm abnormalities was still under the values found for the Kenney extender by day three. Ferreira et al. (1991) also showed that donkey sperm motility of cooled semen was better preserved in extender based on egg yolk than in skimmed milk extender. It was concluded that for in vitro donkey sperm preservation at 5° C, splitting the rich seminal fraction did not improve longevity and preservation of sperm integrity, and that modified Baken extender protected better sperm viability than Kenney extender. ACKNOWLEDGEMENTS This research was financially supported by the FAPEMIG and we thank donkey breeders for their help. AURICH, J.E., KUHNE, A, HOPPE, H. et al. Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation. Theriogenology, v.46, p.791-797, 1996. 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