Semen cryopreservation is an important section of artificial insemination programs (
Zinc is one of the important trace elements in the body, while its deficiency causes infertility in most animals due to disorders of testes development, spermatogenesis (
Zinc ions affect the expression of some germ-cell specific-genes during the course of spermatogenesis in sheep (
Information on the
In this prospective longitudinal laboratory study, semen samples of 5 buffalo bulls of 3-5 years old kept in Buffalo Breeding Center of North-West of Iran, Urmia (37° 33' N, 45° 4' E), were collected by artificial vagina at 5 different occasions at weekly intervals during the late summer and autumn 2011. A total number of 25 samples were used in each examination. Semen samples were immediately transferred into a 37℃ incubated at 37˚C for 40 minutes. Aftl (appearance, homogeneity, density, color and volume) examination. Sperm gross and progressive motility, viability, and abnormal morphology of semen were evaluateded. Thin, watery specimes with low quality were discarded. These parameters (except gross motility) were measured within 0.5(T0), 1(T1) and 2(T2) hours after diluting semen (1:10 v/v) in the Tris-citric acid extender traditioallyused in the center [(without egg yolk and glycerol), pH=7.1, osmotic pressure≡300 mosmolkg-1 and all the chemicals were purchased from Merck Co., Germany]. The extender consisting of Tris 2.66 g, glucose 1.2 g, citric acid 1.39 g, cysteine 0.139 g and distilled water in total volume of 100 ml that contained none (control, without zinc sulphate), 0.072, 0.144, 0.288, 0.576 and 1.152 mg/L zinc sulphate (ZnSO4, 7H2O, ScharlauChemie S.A., Sentimental, Spain). Next, a Tris-citric acid-egg yolk-glycerol extender (above mentioned extender with 20% egg yolk and 7% glycerol, added in one step, as protectant and without antimicrobial agents) containing the same amounts of zinc sulphate was prepared at room temperature. The semen samples were diluted at a rate of 1:10 v/v, (an approximate count of 12×106 sperm cells/mL), cooled to 4℃ within 2 hours, transferred to the equilibrium chamber of 4℃ and left for 4 extra hours to equilibrate. Sperm parameters (progressive motility, viability, membrane integrity and DNA damage) were estimated in equilibrated semen and semen was then loaded in 0.5 ml French straws and frozen over liquid nitrogen in static vapor up to .120℃ within 25 minutes before being plunged in the liquid nitrogen (
Sperm progressive motility was estimated using a computer assisted system of analysis (CASA) [Hoshmand Fannavar (HF) CASA, version 6, Amirkabir Medical Engineering Co, Tehran]. After the samples were stained using eosin-nigrosin staining method (
Hypo-osmotic swelling test (HOST) was used to examine membrane integrity of spermatozoa before and after freezing according to a method of Jeyendran et al. (
DNA damage was detected using acridine orange staining technique, according to the method of Katayose et al. (
TAC of the frozen-thawed semen was assayed in duplicates using a commercial kit (Antioxidant Capacity Assay Kit, Cayman Chemical Co., Ann Arbor, MI, USA).
The data obtained from 25 semen samples from 5 bulls (total number of 125 assays for each estimation) were analyzed using SAS (Version 9.2, SAS Institute Inc., Cary NC.). Prior to analyses, data were checked using box plots to examine for errors and outliers. No outlier was detected. The parameters were analyzed using a linear mixed model (PROC MIXED) with the REPEATED command. The Kenward-Roger procedure was used to approximate the denominator degrees of freedom. The residuals were assessed visually by quantile-quantile plots for testing of the normality and also predicted values were plotted versus residuals to assess the homogeneity of variance. To meet the assumptions of the tests, the data for all parameters, except than that for TAC, were transformed by applying square root transformation. The initial model for each parameter included treatment, stage and their interactions as fixed effects and buffaloes nested in treatment were considered as random effect. When a non-significant interaction term was detected, the model was re-run with the interaction effect excluded from the model. For each parameter several covariance structure between repeated measures were examined and the model which provided better fit according to minimum Akaike’s Information Criterion (AIC) was used. Differences between least squares means were determined using the DIFF option and Bonferroni’s correction was applied to pair-wise comparisons. All reported values are least squares means and statistical mean and standard error of mean (SEM). Effects were declared significant at p<0.05. This study was approved by the Ethic Committee of Urmia University.
The results are summarized in tables 1 and 2. Adding 0.288 mg/L zinc sulphate to the extender preserved the sperm motility at T1 and T2 compared to controls (85.4 ± 1.2% vs. 84.8 ± 1.5% at T0, 85.5 ± 1.1% vs. 80.8 ± 1.3% at T1 and 85.7 ± 1.1% vs. 77.4 ± 1.5% at T2) which was significant (p<0.05) at T1 and T2 indicating that the highest value belongs to only at T2, whereas shows a significant effect on sperm viability (86.3 ± 0.9%, p<0.05,
In equilibrated diluted semen the highest sperm progressive motility (80.7 ± 1.9%), viability (84.1 ± 1.0%) and sperm membrane integrity (83.3 ± 0.9%) values were observed in 0.288 mg/L zinc sulphate, the number of intact DNA cells (green cells) in this diluted solution was not statistically different from control. Adding 1.152 mg/L zinc sulphate to the extender, however, significantly (p<0.001) reduced motility, viability and membrane integrity while increasing sperm cell DNA damagerate (
Effects of different zinc sulphate concentrations (mg/L) on the motility and viability (LS mean ± SEM) of spermatozoa in freshly diluted semen
|T0||84.8± 1.5ab||84.2± 1.4ab||85.0± 1.8ab||85.4± 1.2a||80.7± 1.7b||75.2± 1.2c||0.0025||<0.0001||<0.0001||<0.0001|
|T1||80.8± 1.3ac||81.0± 1.4ac||83.5± 1.6ab||85.5± 1.1b||78.2± 1.7c||71.7± 1.2d||0.0025|
|T2||77.4± 1.5a**||78.9± 1.2bc**||81.6± 1.4b||85.7± 1.1c||72.7± 1.0d***+||67.9± 1.2e***+||0.0025|
|T0||86.1± 1.2ab||86.3± 1.2ab||87.3± 1.5a||87.2± 1.0ab||83.5± 1.6b||77.8± 1.2c||0.0016||<0.0001||<0.0001||0.0220|
|T1||83.8± 1.5ab||84.2± 1.2a||85.6± 1.4a||86.8± 1.0a||80.2± 1.6b||74.0± 1.3c*||0.0016|
|T2||81.7± 1.1ac*||80.2± 1.4ac**+||83.5± 1.2ab||86.3± 0.9b||78.7± 1.2c*||71.7± 0.9d***||0.0016|
T0; 0, T1; 60, T2; 120 minutes after diluting semen, a, b, c, d, e ; Signify a significant difference (p<0.05) within the same row, *; Signifies a significant difference (p<0.05) with T0 values, **; Signifies a significant difference (p<0.01) with T0 values,***; Signifies a significant difference (p<0.001) with T0 values, +; Denotes a significant difference (p<0.05) with T1 values, LS; Least square, TRT; Treatment and STG; Stage.
Effect of different zinc sulphate concentrations (mg/L) on sperm parameters (LS mean ± SEM) after equilibrium and thawing* time
|EQ||71.4± 1.9a||73.5± 1.8ab||78.3± 1.6bc||80.7± 1.9c||64.1± 1.6d||56.9± 1.3e||0.0081||<0.0001||<0.0001||<0.0001|
|TW||40.5± 1.7a||43.2± 2.0ab||47.8± 1.9b||53.7± 1.8c||32.9± 1.7d||27.6± 1.6e||0.0081|
|EQ||78.0± 1.5a||79.1± 1.3a||80.4± 1.2a||84.1± 1.0b||77.4± 1.1a||73.5± 1.3c||0.0036||<0.0001||<0.0001||<0.0001|
|TW||60.1± 1.5a||62.0± 1.6ab||65.7± 1.8b||70.8± 1.8c||59.6± 1.8a||53.7± 1.9d||0.0036|
|EQ||79.0± 1.2ac||79.4± 1.3ac||81.7± 0.9ab||83.3± 0.9b||77.8± 1.1c||73.8± 1.5d||0.0036||<0.0001||<0.0001||<0.0001|
|TW||56.6± 1.7a||58.9± 1.7a||62.1± 1.8b||67.3± 1.6c||54.9± 1.7a||48.8± 2.2d||0.0036|
|EQ||3.0 ± 0.25a||3.4 ± 0.21a||3.4 ± 0.23a||3.0 ± 0.25a||3.5 ± 0.24ab||4.14± 0.21b||0.0025|
|TW||11.8 ± 0.33a||11.6 ± 0.43a||10.3± 0.49b||10.1± 0.47b||12.2± 0.33ac||13.3± 0.26c||0.0025||<0.0001||<0.0001||0.3084|
|TW||63 ± 3.2a||64 ± 3.7a||77 ± 3.4b||81 ± 3.3b||57 ± 2.7ac||49 ± 2.4c|
*; 30 seconds in a 37˚C water bath, EQ; After equilibrium, TW; After thawing, TAC; Total antioxidant capacity, a, b, c, d, e; Signify a significant difference (p<0.05) within the same row, LS; Least square, TRT; Treatment and STG; Stage.
In frozen-thawed semen, the highest sperm motility (53.7 ± 1.8%, p=0.000), viability (70.8 ± 1.8 %, p=0.001) and sperm membrane integrity (67.3 ± 1.6%, p<0.05) values, were observed in 0.288 mg/L zinc sulphate, indicating that this concentration is significantly higher than the other treatments. Furthermore, the least number of DNA damaged cells (p<0.01) was obtained in this treatment (
Sperm motility and fertility is reduced by sperm processing and cryopreservation involved in the semen preservation in artificial insemination programs (
The extender used in this study was Tris-citric acid base extender that had been used in the Buffalo Breeding Center for some years but the egg yolk, glycerol and antibiotics were not added to it in the first stage, in order not to interfere with the examination and in the view that they might have some effect on the sperm parameters. Andrabi (
In this study a CASA was used for the sperm progressive motility evaluation, but since the system was not calibrated for buffalo sperm, progressive motility of 25 semen samples were examined visually coincident with the system evaluation and the results were compared. The difference of two readings was less than 3%. So, only this parameter of motility was used in the study. The other parameters of motility were not used because their reliability for buffalo semen was not assured.
Sperm progressive motility, viability and abnormal morphology were evaluated immediately after the semen being diluted with the extender containing different dozes of zinc sulphate at different time intervals in order to assess the dose and time effect of zinc sulphate on spermatozoa quality. In all the examinations, all the treated samples were compared with their own control which had no zinc sulphate in its extender.
In selecting zinc sulphate dose two points were considered: 1. molecular weight (MW) of "zinc sulphate, 7H2O" is nearly 288 (287.57) grams with 65 gram zinc content, 2. Storage media, commercially prepared for
The present study revealed that supplementation of zinc sulphate improved the quality of freshly diluted and frozen semen of buffalo bulls as compared to the non-supplemented control group. An earlier study on buffalo semen by Alavi-Shoushtari et al. (
As most of sperm functions originated from the membrane, zinc may improve sperm functional capacity by creating a favorable environment (
A low percentage of motile sperms after adding high levels of zinc sulphate (0.576 and 1.152 mg/L) to the extender observed in this study could be due to an elevated free zinc fraction and its subsequent uptake by spermatozoa (
In this study zinc is believed to be important for membrane and chromatin stability and sperm motility (
Storage time affected quality of the freshly diluted semen by deteriorating sperm progressive motility and viability which was more evident in the control group and low doses of zinc sulphatesupplementation groups, while adding 0.288 mg/ml zinc sulphate to the extender showed significantly better result. In equilibrated semen some spermatozoa did not tolerate the process. This was evident by lower semen quality (
The results showed that 0.288 mg/L zinc sulphate improve to the sperm quality (progressive motility, viability, membrane integrity and total antioxidant capacity) preservation upon freezing processes, however, we suggest a bigger sample population to achieve a definite statement. Our finding also revealed that zinc affects the cell membrane and leads to a lower degree of sperm DNA damage after semen freeze-thawing, which in turn, results in higher semen fertility. However, addition of higher zinc concentrations (0.576 and 1.152 mg/L) are detrimental to spermatozoa.