SUMMARY: Pisum sativum lectin (PSA) has been used successfully as a marker of glycoconjugates located on the inner acrosomal membrane, but due to changes in the constituents of membranes between species, their use requires a preliminary validation. This study compares the results of acrosomal staining with a combination of propidium iodide (PI) and PSA with Rose Bengal stain that is commonly used as standard, and also compares the interpretation of staining results obtained with PI / PSA by two independent evaluators.
In experiment 1, goat semen was collected by artificial vagina, washing and dyeing of seminal plasma in comparison with PI / PSA and Rose Bengal, initially directly after collection and then after preparing fractions with different proportions of sperm living and dead. In experiment 2, frozen semen from different goats at -196 ^0 C in Tris-based diluent, citrate and egg yolk was thawed at 37 ^0 C for 30 sec or at 65 ^0 C for 5 sec and the sperm were stained with PI / PSA. The interpretation of the results the acrosome integrity was performed by two independent evaluators. To compare the results the percentages were transformed and the means and correlations were calculated using the statistical package Systat (R).
The results show that the stain PI / PSA and Rose Bengal gives similar results and positively correlated (r = 0.99) and the interpretation of the evaluators was also positively correlated (r = 0.98). According to the above, we conclude that the staining PI / PSA is useful for rapid assessment of acrosome integrity in goat.
SUMMARY: Use of propidium IOID and Pisum sativum for fast Assessment of acrosome
integrity in goat spermatozoa
Lectins Are Used For studies in mammalian sperm surface and organelles Because of Their Ability to specifically link residual sugar. Fluorescein labeled Pisum sativum (PSA) has Been Used in Several mammalian spermatozoa successfully, as a marker of the inner acrosomal membrane But, Because of interspecific Variations in membrane Constituents, ITS use Requires a preliminary validation. This study Compare The results Obtained goat spermatozoa by staining with a combination of propidium IOID (PI) and PSA vs Bengal Network of Those Also the interpretation of the results Were Achieved Compared with the results Obtained by two external Technicians.
In Experiment 1, goat spermatozoa collected by artificial vagina Were washed eleven Immediately after collection, and stained with PI / PSA and Rose Bengal. Then, to prepare sperm groups with Different Proportions of live and dead sperm, spermatozoa and one fraction fractionated Were WAS damage by freezing at -196 ^0 C Without cryoprotectans. In Experiment 2, semen from Different evils WAS frozen in a Tris-base d extend, and Either thawed at 37 ^0 C for 30 sec or at 65 ^0 C for 5 sec, And Then stained with PI / PSA. Two independent Technicians Assessed the results of Both series of experiments. Percentage Transformations, mean comparison and correlation Were Obtained by computing using the Systat (R) package.
The results Eichmann That PI / PSA and Rose Bengal and Positively Correlated Gave similar results and similar Gave That Both Technicians Interpretations (r = 0.98). It Can Be Concluded That PI / PSA staining for SP, is a fast Assessment of goat spermatozoa.
Keywords: sperm, lectins, acrosome, goats.
Key words: spermatozoa, lectins, acrosome, goats.
The sperm acrosome is an organelle that plays an essential role in the fertilization process. It is covered externally by the plasmalemma and outer acrosomal membrane and internally by the inner acrosomal membrane (Bedford, 1990). The functional significance of the acrosome is due to its role in setting and sperm penetration of zona pellucida and the fusion of gametes (Yanagimachi, 1988). Although the inner acrosomal membrane is tough enough, the double membrane that forms the acrosomal wall can easily be altered physiological effects (eg acrosome reaction) or physiological (eg, degeneration, injury physio).
The evaluation of acrosome integrity was important, both from a clinical standpoint, technological and research from biology sperm (Bavister, 1990; Cross and Meizel, 1989), so that in species with a relatively small acrosome noticeable, as in domestic ruminants, have developed various techniques to facilitate their identification.
Bearing in mind the variations between species, the standard staining procedures have proven quite useful, but in turn are laborious, time demanding and sometimes their results are somewhat ambiguous (Cross and Meizel, 1989).
Recently incorporated the use of lectins associated with fluorescein as a marker of the acrosome and plasma changes in ejaculated sperm (Cummins, 1995, Medeiros and Parrish, 1996). Lectins are glycoproteins that specifically bind to terminal sugars located in cellular structures and, therefore, have been used as specific markers of glycoconjugates localized in both acrosome intact and in the acrosomal matrix (Cross et al., 1986; Nolan et al., 1992, Way et al., 1995). The lectin Pisum sativum (PSA) can recognize terminal mannose, glucose and glucosamine (Goldstein and Portez, 1986) of glycoconjugates located on the acrosomal matrix and therefore associated with a fluorescein can identify sperm with damaged or missing acrosome (Nolan et al ., 1992, Aitken and Brindle, 1993). Propidium iodide (PI) is a nuclear stain and therefore serves as a sperm stain contrast. In combination, they allow a rapid assessment, simple and unambiguously to the presence or absence of acrosomal sac.
In general, the ideal sperm tests are simple, quick to perform, reliable, repeatable and should provide relevant information (Bavister, 1990). The great usefulness of lectins in the functional assessment of spermatozoa is fulfilling most of these requirements (Kawakami et al., 1993), at which have been used increasingly in studies of sperm biology.
As a marker of acrosomal function, the lectin PSA has been used successfully in bulls (Nolan et al., 1992, Way et al., 1995), foals (Farlin et al., 1992, Casey et al., 1993) and dogs (Kawakami et al., 1993, Gonzalez-Chabarri et al., 1994), while in many human clinical trials have begun to be used in conjunction with the lectin Arachis hypogea (PNA) as a technique of choice for evaluating acrosomal (King and Rajalakshmi, 1995). The function of this paper is to describe the use of the combination of PI and PSA adjusted for evaluation of acrosome integrity in goat.Material and methods
Processing and freezing of semen. Semen from 4 cookies Anglo Nubian sexually mature was collected by artificial vagina, suspended in TALP medium without calcium (with 10% v / v goat serum, Parrish et al, 1988) and washed by centrifugation at 200 g for 5 min and resuspension of sperm in the same medium, and evaluated on the basis of the progressive movement in phase contrast microscopy.
The freezing of semen was carried out according to Evans and Maxwell (1987). In summary, the semen was diluted in a diluent consisting of Tris (4543 g), glucose (0.750 g), citric acid (2.606 g), egg yolk (3.0 ml), glycerol (6.0 ml), penicillin (100,000 IU) , streptomycin (100.0 mg) and distilled water (100 ml) and allowed to reach laboratory temperature (-20 ^0 C). Sperm concentration was adjusted to 240 x 106 sperm / ml and the semen was stored in sterile 0.25 ml straws (IMV, France). Subsequently, semen was cooled to 5 ^0 C at a rate of 1 ^0 C / min and frozen and maintained in liquid N 2 vapor for 10 min. Subsequently, semen was deposited directly in liquid N2 and stored in portapajuelas identified.
Sperm stains. Standard acrosome stain to contrast the use of lectin Rose Bengal staining (Talbot and Chacon, 1981), with some adjustments for goat sperm, and according to the criteria of semen handling and described by Perez-Llano Pintado (1992). In summary, sperm were fixed for 20 min at 37 ^0 C in glutaraldehyde (2%) diluted in sodium cacodylate buffer (0.1M, pH 7.4), then washed in deionized water 2 times and then 2 smears were prepared were air dried. The smears were stained for 15 min with Rose Bengal to 0.8% in Tris buffer (pH 5.3) and then washed with water, dehydrated in a battery of alcohol, clarified with xylene, sealed with Entellan and covered with coverslip. The assessment was made in light microscopy with an objective of x 100. Staining clearly demarcates the acrosomal ridge in the presence of intact acrosomes, so the absence of such a ridge was evaluated as evidence of an acrosome absent.
PSA lectin conjugated with fluorescein isothiocyanate (FITC, 2.5 mol FITC / mol of PSA) and PI were prepared in a stock solution of 0.1 and 0.125 mg of lectin / ml of phosphate buffer (PBS) respectively, and stored at -20 ^0 C, protected from light. To assess the integrity of acrosome, sperm concentration was adjusted to 50 x 106 sperm / ml in a fixative solution (5 cc of formalin to 40% / It of sodium citrate solution 2.9%) and incubated for 10 min at 39 ^0 C. Subsequently, 0.2 ml of sperm suspension was incubated at least 30 min with 16 ul of PSA and 8 ul of IP, and then 30 ul were deposited on a clean slide and covered with a coverslip to facilitate its evaluation. The evaluation was performed under an objective of x 40 using a Nikon fluorescence microscope. PI stains all red sperm through membrane permeabilization carried out by formaldehyde, while the PSA stained with a yellowish-green region of the sperm acrosome only with damaged or missing acrosome.
The chemical and biological agents were obtained from Sigma Chemical Corp (St. Louis, MO, USA), unless otherwise indicated.
Experiment 1. Correlation between the assessment of acrosome integrity measured by lectin and Rose Bengal. In the first part of the experiment, semen from 4 goats were washed, incubated and then acrosome integrity was directly assessed. In the second part, previously washed semen of the same males was divided into two parts: the first was frozen without cryoprotectants, depositing the semen stored in cryovials directly into liquid N2, while the second, kept unaltered, was used to in combination with the previous prepare 3 to assess sperm fractions: a fraction of a normal, partially damaged (50% and 50% normal thawed) and a damaged portion (100% of thawed sperm) for the freezing process. Acrosome integrity of at least 200 spermatozoa was evaluated by PI / PSA and Rose Bengal, the first in duplicate for analysis by two trained, independent evaluators.
Experiment 2. Assessment of acrosome integrity in response to different thawing temperatures. Frozen semen stored in 0.25 ml French straws (IMV, LAigle, France) was thawed in a water bath according to the following temperatures and times: a) 65 ^0 C for 5 sec, and b) 36 ^0 C for 30 sec. Subsequently, semen was fixed and stained with PI / PSA and acrosome integrity verified by at least 200 sperm by two independent evaluators.
Statistical analysis. The percentages were transformed by the method of Bliss and then calculated correlations and means were compared to the test of Tukey multiple comparison using the statistical program Systat (R).
The combination PI / PSA stained the acrosomes according to Figure 1. The PI stained spermatozoa to unambiguously a red-orange color that contrasts with the yellowish green color of the lectin labeled with fluorescein isothiocyanate (FITC).
Experiment 1 was designed to test whether the staining patterns obtained with PI / PSA staining were equivalent to the standard and if the interpretation of results between independent evaluators were unequivocal. The experiment results are shown in Tables 1 and 2 for the first and second parts, respectively. The results of the first and second part of experiment 1 show that the information obtained with PI staining / PSA is similar to that obtained with Rose Bengal. Moreover, the interpretation of the results by independent evaluators has a highly significant positive correlation.
Figure 1. Staining of sperm with a combination of PI and PSA. The orange-red color corresponds to the fluorescence emitted by PI, while the yellowish green color corresponds to FITC-conjugated lectin PSA (magnification x 1000).
Sperm staining with PI / PSA (1000 x). Whereas network Intact stain spermatozoa spermatozoa with acrosome Damaged yellow-green stain.
Evaluation of the repeatability of the measurement of acrosome integrity using PI / PSA
sperms, according to the comparison with Rose Bengal stain
and with different evaluators.
Evaluation of repetition of acrosome Assessment using PI / PSA in ejaculated spermatozoa
Compare with Bengal as Red staining.
Evaluation of the repeatability of the measurement of acrosome integrity using PI / PSA
sperm populations treated with cold, according to the comparison with staining
Rose Bengal and other evaluators.
Evaluation of the repetition of the acrosome Assessment using PI / PSA in cold-treated spermatozoa,
Compare with Bengal as Red staining.
In experiment 2 staining was used to assess acrosomal damage produced by the method of thawing semen and correlated the results obtained by two independent evaluators. The results are shown in Table 3.
The results show no significant effect of semen thaw method on the integrity of acrosomes (p> 0.05). Also, in this experiment, the interpretation of the results by independent evaluators made a highly significant positive correlation.
In this study, the combination PI / PSA proved to be reliable, according to the equivalence of results with Rose Bengal staining, and demonstrated to allow unambiguous interpretations, according to the correlation of the results obtained by independent evaluators. Using formaldehyde as a fixative citrate, there was no clumping problems described for bull sperm with heparin trained Way et al. (1995), although agglutination was seen in glutaraldehyde-fixed sperm (results not shown). The above results suggest that combining PI / PSA can be used for rapid assessment of acrosome integrity in goat spermatozoa.
Evaluation of the repeatability of the measurement of acrosome integrity using PI / PSA according to the effect of thawing on acrosomal structure.
Evaluation of the repetition of the acrosome Assessment using PI / PSA, as Affected by the Thawing procedure.
The authors thank Dr. M. Briones by statistical analysis of results.
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Depto. Animal Science, Faculty of Veterinary Medicine, University of Concepcion, Casilla 537, Chillan, Chile.