Papers & Articles Relationship between the nuclear morphology of the sperm of 10 bulls and their fertility A. L. Vieytes, H. O. Cisale, M. R. Ferrari The relationships between the fertility and nuclear morphology, chromatin maturity and chromatin condensation of the sperm of three bulls with a calving rate over a year of more than 65 per cent, four bulls with a calving rate between 65 per cent and 35 per cent, and three bulls with a calving rate of less than 35 per cent were studied. The sperm nuclei were stained with the Feulgen reaction, and chromatin condensation and maturation were evaluated in situ by staining with toluidine blue and acid aniline blue. Nuclear chromatin decondensation was induced with dithiothreitol; this showed that in the bulls with low fertility, more than 35 per cent of nuclei were decondensed, and that one of them had the lowest percentage of normal nuclei (64·9 per cent) and stronger positive reactions to the acid aniline blue and toluidine blue stains than the other bulls. Veterinary Record (2008) 163, 625-629 A. L. Vieytes, MSc, Área Producción de Bovinos de Leche, H. O. Cisale, PhD, M. R. Ferrari, PhD, Área Fisica Biológica, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Chorroarin 280, CP 1427, Ciudad Autónoma de Buenos Aires, Argentina Correspondence to Dr Ferrari THE most accurate method for testing the fertility of frozen bull semen is the insemination of many fertile females, but this method takes too long and is too expensive for routine use (Barth and Oko 1989). The development of simple, inexpensive laboratory tests that could accurately predict the fertility of a bull or preserved semen samples has been the goal of many research programmes. The routine tests used to predict fertility are measurements of sperm concentration, percentage motility, percentage viability and morphology, but they may not reveal subtle sperm defects (Hammadeh and others 2001, Evenson and others 2002, Sharma and others 2004). In this context, the integrity of the sperm chromatin and its degree of condensation are important measurements for the prediction of fertility (Evenson and others 2002, Erenpreisa and others 2003, Sharma and others 2004, Enciso and others 2006). The nuclear chromatin of mammalian sperm has a peculiar organisational status characterised by a remarkable process of remodelling or condensation (Govin and others 2004, Caron and others 2005). The sperm DNA is organised in a specific way that keeps the chromatin compact and stable in the nucleus. It is packed into a tight, almost crystalline condition and occupies nearly the whole nucleus (Fuentes-Mascorro and others 2000). This process of condensation occurs in two main phases. The first phase, which occurs in the testis, involves the substitution of somatic histones by testis-specific protamines (Caron and others 2005). The protamines contain numerous cysteine residues, which generate disulphide cross-links between adjacent protamine molecules during the condensation of the chromatin. The formation of large numbers of disulphide cross-links between protamine molecules occurs in the second main phase of chromatin condensation, when the sperm have left the caput epididymis and are en route to the cauda epididymis (Caron and others 2005). Several methods have been developed to evaluate the maturity of sperm chromatin and DNA integrity (Evenson and others 2002, Agarwal and Said 2003, Sharma and others 2004, Silva and Gadella 2006). The sperm chromatin structure assay (SCSA), which is considered the most efficient and successful assay for evaluating men’s potential fertility, uses flow cytometric analysis to evaluate the fluorescence of sperm stained with acridine orange (Evenson and others 2002). TUNEL and Comet assays are also used to measure DNA fragmentation (Duty and others 2002, Sakkas and others 2002, Evenson and Wixon 2006). However, the relatively high cost of such methods restricts their use, particularly in clinical veterinary practice (Beletti and Mello 2004). Simple staining techniques, such as acid aniline blue and toluidine blue, are easy and inexpenThe Veterinary Record, November 22, 2008 sive methods for the evaluation of some chromatin abnormalities (Hammadeh and others 1996, Beletti and Mello 2004). Aniline blue staining provides a specific positive reaction for lysine. Histone-rich nuclei of immature sperm are rich in lysine and consequently take up the blue stain. Protaminerich nuclei of mature sperm are rich in arginine and cysteine and contain relatively low levels of lysine, which means they are not stained by aniline blue (Dadoune and others 1988, Hammadeh and others 1996). Toluidine blue is a basic nuclear dye used for chromatin, metachromatic and orthochromatic staining (Stockert and others 1991, Erenpreisa and others 2003, Beletti and Mello 2004); sperm with chromatin abnormalities are usually stained from dark blue to violet, whereas normal sperm are pale blue or not stained. The use of disulphide bond-reducing agents allows the percentage of immature sperm present in ejaculates to be evaluated (Liu and others 1987). In several studies, nuclear decondensation has been induced to analyse the degree of chromatin maturation and its stability (Zirkin and others 1985, Liu and others 1987, Madrid-Bury and others 2005, Enciso and others 2006). Furthermore, sperm with chromatin abnormalities frequently have abnormally shaped heads and have been associated with reduced fertility or abortions (Chemes and Rawe 2003). Several studies have observed relationships between chromatin structure, DNA damage and abnormal morphology in bull sperm (Ballachey and others 1988, Sailer and others 1996, Karabinus and others 1997, Ferrari and others 1998, Ostermeier and others 2001). The Feulgen reaction, a staining technique, is both specific and stoichiometric for nuclear DNA, and makes it possible to observe the nuclear morphology of sperm and the abnormally condensed chromatin. Dobrinski and others (1994) observed significant correlations between observations of Feulgen-stained nuclei and the results of the SCSA test. The aim of this study was to relate the results of simple, inexpensive, practical and accurate laboratory tests with the fertility of bulls used for artificial insemination (AI). MATERIALS AND METHODS Frozen semen from 10 bulls was obtained from AI centres. More than 65 per cent of the cows inseminated by three of them during a year had calved, between 65 per cent and 35 per cent of the cows inseminated by four had calved, and less than 35 per cent of the cows inseminated by the other three had calved. These calving rates were determined from the Papers & Articles number of births resulting from at least 1000 inseminations per bull. The exact number of ejaculates used in the calculation of the calving rates was not provided. Cryopreserved sperm was used in all cases; the straw size was 0·5 ml and a commercial extender was used. Each sample was thawed at 37°C for 30 seconds and diluted to 106 sperm/ml. Two straws from the same ejaculate from each bull were analysed. Aniline blue staining Air-dried smears were fixed in 3 per cent glutaraldehyde in phosphate-buffered saline (PBS) for 30 minutes, dipped twice in PBS for five minutes, stained with 5 per cent aniline blue at pH 3·5 for seven minutes, washed with PBS and air dried (Dadoune and others 1988). Toluidine blue staining Air-dried smears were fixed with methanol for two minutes, stained with 0·2 mg/ml toluidine blue for two minutes, washed briefly in distilled water and air dried (Barrera and others 1993). Feulgen reaction Air-dried smears were fixed in ethanol-acetic acid (3:1, v/v) for 30 minutes and then washed three times for 10 minutes, each time with distilled water. The smears were hydrolysed for 40 minutes in 5N hydrochloric acid at 22°C, washed three times for 10 minutes each time with distilled water and stained with Schiff’s reagent at pH 2·2 for 60 minutes in darkness. The stained material was washed three times for 10 minutes each time in sulphurous water (180 ml distilled water plus 1 ml 10 per cent sodium bisulphite and 10 ml 1N hydrochloric acid at 5°C in darkness (Ferrari and others 1996). Normal and abnormal nuclear morphologies were described according to Barth and Oko (1989). Nuclear chromatin decondensation One volume of semen was mixed with one volume of 1 per cent aqueous dithiothreitol (DTT) at pH 4·5 and incubated at room temperature for 11 minutes, as described by Vieytes and others (2005). The reaction was stopped by smearing the sperm suspension onto glass slides, followed by air drying (Barrera and others 1993) and staining with the Feulgen method as above. The smears were evaluated under brightfield microscopy at 1000 x magnification. Statistical analysis Descriptive statistics were used and the differences between individual bulls were tested by chi-squared tests. TABLE 1: Percentages of nuclei with different morphologies observed on 500 Feulgenstained sperm cells from each of 10 bulls Nuclear sperm morphology (%) Normal Vacuolated Pyriform With crest Large Round With abnormal condensation Small Tapered Diploid Fertility Bull 1a 2b 3c 4b 5b 6b 7b 8d 9b 10a 82·7 5·3 5·7 0·6 0·0 0·0 93·1 0·7 1·8 0·0 0·9 0·5 96·3 0·0 0·3 0·16 0·2 0·0 92·3 0·0 2·0 0·0 0·0 0·1 92·8 0·0 1·8 0·0 0·0 0·0 93·9 1·1 1·7 0·0 1·2 0·6 93·9 0·7 1·0 0·0 2·1 0·0 64·9 12·5 9·3 1·2 1·0 0·8 90·0 5·5 1·8 1·0 0·0 0·6 79·8 7·6 5·9 0·0 1·5 0·8 0·0 0·6 4·7 0·4 H 0·0 1·4 1·4 0·0 H 0·0 1·1 0·6 0·0 H 0·6 2·6 2·3 0·0 M 0·7 2·7 1·9 0·0 M 0·0 0·6 0·8 0·0 M 1·1 0·5 0·5 0·2 M 0·8 5·9 2·2 1·5 L 0·0 0·0 0·0 0·0 L 1·3 2·5 0·0 0·0 L Different subscript letters denote significant differences (P<0·05) between bulls in the proportion of normal/abnormal nuclei H High, M Medium, L Low Table 2 also shows the percentages of sperm stained with toluidine blue. Sperm from bulls 3, 4, 5, 6 and 9 were not stained, 6 per cent of the sperm cells from bull 8 were intensely stained, and bulls 1, 2, 7 and 10 had significantly lower percentages of stained sperm. The Feulgen reaction showed that the exposure of sperm to DTT modified the distribution of chromatin. To facilitate evaluation of these changes, four nuclear categories were established, as described by Vieytes and others (2005): type 1 (no reaction or no observable changes under brightfield microscopy), type II (a smaller than normal nucleus, intensely stained), type III (a vacuolated nucleus with some loss of shape) and type IV (a very decondensed nucleus, with relatively large vacuoles and significant loss of shape). Fig 1 shows the different types of nuclei observed, and Table 3 shows the percentages observed in the 10 bulls. Types III and IV were difficult to distinguish, and they were combined into one category (III+IV) containing all the nuclei in which the sperm chromatin was decondensed when observed under brightfield microscopy. The sperm of bull 8 had the highest proportion of sperm with decondensed chromatin induced by DTT. Bulls 9 and 10, the other two bulls with low fertility, also had higher proportions of decondensed nuclei than the other seven bulls with high or medium fertility. Fig 2 shows the responses to DTT TABLE 2: Percentages of 1000 sperm nuclei, from each of 10 bulls, that stained with aniline blue (AB) and toluidine blue (TB) Bull RESULTS Several types of abnormal nuclei were recognised: vacuolated, pyriform, crested, large, round, with abnormal condensation, small, tapered and diploid. Table 1 shows the percentages of the different morphologies observed. The samples from seven of the 10 bulls had more than 90 per cent of normal sperm nuclei. The relationship between the number of normal and abnormal sperm nuclei did not differ significantly between bulls 2, 4, 5, 6, 7 and 9, or between bulls 1 and 10. Bull 3 had the highest percentage of normal nuclei (96·3 per cent) and bull 8 the lowest (64·9 per cent); they differed significantly from each other and from the other eight bulls (P<0·05). Bull 8, which had the lowest fertility, had high percentages of vacuolated (12·5 per cent), pyriform (9·3 per cent) and small (5·9 per cent) nuclei (Table 1). The results of the observations on sperm stained with acid aniline blue are shown in Table 2. Bull 8 showed a response to this stain that differentiated it from the other bulls. Stain AB TB Fertility 1 2 3 4 5 6 7 8 9 10 0·0 0·2 H 2·6 0·2 H 6·5 0·0 H 1·5 0·0 M 1·6 0·0 M ND 0·0 0·2 M 100 6·0 L ND 4·2 0·2 L 0·0 M 0·0 L ND No data, H High, M Medium, L Low TABLE 3: Percentages of the nuclei of 500 sperm from each of 10 bulls that showed different reactions to exposure to 0·5 per cent dithiothreitol for 11 minutes, as observed on Feulgenstained nuclei Bull Nuclear categories I II III+IV Fertility 1 2 3 4 5 6 7 8 9 10 0·0 8·0 15·6 H 1·9 3·6 11·9 H 5·3 19·6 26·8 H 0·0 11·1 24·9 M 0·7 3·5 31·3 M 5·8 10·2 10·2 M 6·7 0·4 15·9 M 0·0 36·8 53·6 L 36·1 38·8 38·8 L 1·2 17·1 35·3 L I No reaction, II Highly stained small nuclei, III+IV Decondensed nuclei H High, M Medium, L Low The Veterinary Record, November 22, 2008 Papers & Articles II I IV III II I II III IV I IV III III FIG 1: Examples of sperm nuclei illustrating different grades of nuclear reaction observed after the spermatozoa had been exposed to 0·5 per cent dithiothreitol for 11 minutes and the nuclei had been stained with the Feulgen reaction specific for DNA. The grades shown are: I No reaction, II Highly stained small nuclei, III and IV Decondensed nuclei. Bar=10 µm I III I IV IV I treatment of the sperm from the three bulls with low fertility in comparison with those of the bulls with medium and high fertility. DISCUSSION The quality of sperm must be strictly controlled when cryopreserved bull semen is used for AI. One possible cause of infertility or subfertility is a change in the status of the nuclear chromatin, and analysis of chromatin condensation and maturity may therefore be valuable for routine evaluation of cryopreserved bull sperm (Madrid-Bury and others 2005). Associations between disturbances in sperm condensation, nuclear morphology and infertility have been described in many reports by Hammadeh and others (1996) and Chemes and Rawe (2003). In the present study, the nuclear morphology of the sperm of 10 bulls with different levels of fertility was evaluated. Seven of the bulls had up to 90 per cent of sperm nuclei with normal morphology, but three of them had lower percentages. Two of these three also had a low fertility, and the principal defects in their sperm – pyriform nucleus, small nucleus and nuclear vacuoles – have all been The Veterinary Record, November 22, 2008 associated with fertility problems (Barth and Oko 1989). The Feulgen technique proved to be a valuable method for studying sperm nuclear morphology; it has been shown that Feulgen staining is correlated with the results of flow cytometry (SCSA) (Dobrinski and others 1994). The grade of nuclear condensation or maturation of sperm can be assessed by staining with aniline blue, which discriminates between lysine-rich histories and arginine/ cysteine-rich protamines (Dadoune and others 1988). The histone-rich nuclei of immature sperm contain abundant lysine and react positively by taking up the stain, whereas the protamine-rich nuclei of mature sperm contain abundant arginine and cysteine and remain unstained (Hammadeh and others 1996). The results of the present study showed that 100 per cent of the sperm of bull 8, which had poor fertility, were stained by aniline blue, suggesting that this bull had a chromatin maturation problem. In contrast, less than 6·5 per cent of the sperm of the other bulls were stained by aniline blue. Toluidine blue is a nuclear dye used for metachromatic (strong blue) and orthochromatic (slightly blue) staining of chromatin, which is suitable for the assessment of the integrity of sperm cell DNA and chromatin condensation (Barrera and others 1993, Erenpreiss and others 2001, Beletti and Mello 2004). Beletti and Mello (1996) observed that meta- Papers & Articles 65 Decondensed nuclei (%) 55 bull sperm chromatin to decondensation with DTT may be related to differences in their fertility, and that staining with aniline blue and toluidine blue may also be useful for evaluating changes in the nuclear chromatin of sperm. Studies of bulls with a wider range of fertility are needed to assess these tests more thoroughly. 1·96 se 1·00 se Mean 45 35 ACKNOWLEDGEMENTS 25 The authors thank CIAVT and BIOGENETICS Argentina for kindly providing the frozen bull semen used in this study. 15 5 High Medium Low FIG 2: Box and whisker plot of the percentages of decondensed nuclei in the sperm of bulls with high (n=3), medium (n=4) and low (n=3) fertility after treatment with 0·5 per cent dithiothreitol for 11 minutes chromatic staining is more common in subfertile bulls than in highly fertile ones. The present results also suggest that bulls with poor fertility tend to have a higher percentage of metachromatic sperm nuclei. The compactness of the nucleus of sperm is the result of DNA packing due to the formation of many disulphide crosslinks between protamines in the chromatin. It is important to analyse the quality of the condensation of the chromatin because this parameter is directly related to the ability of sperm to fertilise the ovum (Chapman and Michael 2003). Ejaculates from different species contain sperm with various degrees of nuclear stability and resistance to exposure to disulphide bond-reducing agents. The nuclei of immature sperm swell more rapidly and completely than the nuclei of mature sperm (Liu and others 1987). Sperm nuclei are highly resistant to a variety of agents: DTT, EDTA, sodium dodeyl sulphate (SDS), heparin, glutathione, 2-mercaptoethanol, hydrochloric acid, and potassium and sodium hydroxide (Beletti and Mello 1996, Motoishi and others 1996, Madrid-Bury and others 2005). The nuclear chromatin decondensation assay has been performed in different species (Liu and others 1987, Córdova and others 2002), and chromatin decondensation has been obtained in bull sperm using SDS/DTT (Beil and Graves 1977), heparin/DTT (Motoishi and others 1996) and EDTA/SDS (Madrid-Bury and others 2005). The results obtained in the present study using DTT alone showed good chromatin decondensation; the optimal time of incubation with DTT was 11 minutes, as determined by Vieytes (2004) and Vieytes and others (2005). The degree of chromatin condensation varied, but the bulls with low fertility were the only ones that had more than 35 per cent of decondensed nuclei. These results agree with those obtained by Dobrinski and others (1994) and Madrid-Bury and others (2005). Bull 8, which had the lowest chromatin stability index, also had the highest percentages of abnormal nuclei and nuclei stained with toluidine blue and aniline blue. There were low correlations between the percentages of decondensed nuclei and the percentages of other sperm characteristics such as motility (r=–0·15), viability (observed with eosin stain, r=–0·06) and functionally intact membranes (determined with the hypo-osmotic test r=0·40) (Vieytes 2004). The large number of inseminations from each bull should have provided a good estimation of their fertility. Only one ejaculate was used per bull in this study, but it was considered that a single ejaculate would be representative of a bull’s lifetime sperm nuclear characteristics (Ostermeier and others 2001). The results therefore suggest that the response of References AGARWAL, A. & SAID, T. M. 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