**3. Results and discussion**

Our results showed that integrity of plasma membrane is not altered by long‐term storage at −196°CC (**Figures 2A** and **3A**). Specifically, we did not find any significant differences associ‐ ated with the different storage times analyzed (>10 years) or in relation to control (p < 0.05), with a post‐thaw sperm viability percentage that oscillated between 60 ± 1.8 and 68 ± 2.1. A similar situation was observed in the acrosomal integrity analysis, wherein the only significant difference observed associated with storage time was specifically between 45 and 40 years (p  < 0.01) (**Figures 2B** and **3C**). Despite these differences, the percentage of acrosomal integrity after thawing ranged between 87 ± 1.6 and 95 ± 0.5, and ultimately acceptable enough even for fresh semen. On the other hand, comparative analysis of total sperm motility did not show significant differences between times storage analyzed. Specifically, all values shown are above 60%, ranging between 60 ± 2.4 and 66 ± 3.2, similar to viability results (**Figure 2A** and **2C**).

In respect to progressive motility, a higher significant value was found in samples from frozen semen stored by 40 years (p < 0.01) (**Figures 2D** and **3B**). Additionally, a similar behaviour in other cinematic parameters (VSL, VAP, LIN and WOB) was observed in these samples, showing higher values (p < 0.001) (**Table 1**). Consistently, lowest values of hyperactivity, ALH and BCF were observed in the frozen semen stored for 40 years (p < 0.001). This great differences in progressive motility and other cinematic parameters in samples of seminal doses storage by 40 years may have been influenced by the thawing solution used in the pellets tube, particularly with the presence of sodium bicarbonate (30.9 mM) [37].

**Figure 2.** Freezability evaluation of frozen‐thawed bovine sperm. A) Plasma membrane integrity or viability (%) analy‐ sis by CASA system using double stain (propidium iodide and acridine orange). A minimum of 1000 spermatozoa were counted for each assay. B) Acrosomal integrity (%) analysis by Coomassie G‐250 staining. A minimum of 200  spermatozoa were counted for each assay. C) Total sperm motility (%) analysis by CASA System, VCL > 10 μm/s. A minimum of 500 spermatozoa were counted for each assay. D) Progressive sperm motility (%) analysis by CASA Sys‐ tem, STR > 70%. A minimum of 500 spermatozoa were counted for each assay. Each bar (storage time) represents the mean ± SEM of a total of 15 doses were analyzed (from five bulls, three doses for each time analyzed). Significant differ‐ ences among time storing are shown: one‐way ANOVA/Bonferroni post‐test (p < 0.001 or by different letters).

The results shown differ from those obtained by Malik et al. [33], who reported a significant decrease in both the viability and motility associated with prolonged storage (6 years versus 1–2 years). It is highly probable that these discrepancies, in the case of both viability and motility, are due to differences in the sensitivity of the technique used, nigrosin/eosin staining and bright field microscopy versus acridine orange/propidium iodine and epifluorescent microscopy in our case. This could also be due to the evident differences associated with the use of analysis of subjective sperm motility analysis versus our use of CASA system.

There is an argument that spermatozoa store at −79°C (in dry ice) or at −196°C (in liquid nitrogen) retain their fertilizing potential indefinitely [26]; however, the storage time results that studies are controversial. Effectively, although Mazur [29] proposed that several centuries are required, of liquid nitrogen storage, for that ionizing cosmic radiation alters or damages the DNA of the cell. However, there are studies that raise a discrepancy respect that cryopre‐ servation, for several years, completely stops the processes of sperm biochemistry, but whose storage times do not exceed 6 years.

Fourteen five years ago, Salisbury and Hart [16] proposed that bovine frozen sperm have a low fertility level and promote increased embryonic mortality after 1.5 years of storage at −196°C. More recently, Haugan et al. [19] based on results of field trials indicated that the likelihood of conception decreased only a little more than one percentage after 5.5 years of storage, but that level of decline seems to be so important because the calving rate predicted by multiple logistic regression was 59.2%, optimal value according to commercial standard for frozen semen. Contrary, field trial results of Strom [14] found no evidence of reduced fertility when was used frozen semen storage by 1–1.5 years. Additionally, Cassou [17] and Roettger et al [18] reported no difference in fertility when were used frozen semen stored at −196°C for up to 4.5 and 5 years, respectively. Unfortunately, there are no field trials in that and both pregnancy and calving rates have been analyzed; to rule out or confirm effects of prolonged storage on embryo mortality, this would be the only one way to resolve the question.

40 years may have been influenced by the thawing solution used in the pellets tube, particularly

**Figure 2.** Freezability evaluation of frozen‐thawed bovine sperm. A) Plasma membrane integrity or viability (%) analy‐ sis by CASA system using double stain (propidium iodide and acridine orange). A minimum of 1000 spermatozoa were counted for each assay. B) Acrosomal integrity (%) analysis by Coomassie G‐250 staining. A minimum of 200  spermatozoa were counted for each assay. C) Total sperm motility (%) analysis by CASA System, VCL > 10 μm/s. A minimum of 500 spermatozoa were counted for each assay. D) Progressive sperm motility (%) analysis by CASA Sys‐ tem, STR > 70%. A minimum of 500 spermatozoa were counted for each assay. Each bar (storage time) represents the mean ± SEM of a total of 15 doses were analyzed (from five bulls, three doses for each time analyzed). Significant differ‐ ences among time storing are shown: one‐way ANOVA/Bonferroni post‐test (p < 0.001 or by different letters).

The results shown differ from those obtained by Malik et al. [33], who reported a significant decrease in both the viability and motility associated with prolonged storage (6 years versus 1–2 years). It is highly probable that these discrepancies, in the case of both viability and motility, are due to differences in the sensitivity of the technique used, nigrosin/eosin staining and bright field microscopy versus acridine orange/propidium iodine and epifluorescent microscopy in our case. This could also be due to the evident differences associated with the

There is an argument that spermatozoa store at −79°C (in dry ice) or at −196°C (in liquid nitrogen) retain their fertilizing potential indefinitely [26]; however, the storage time results that studies are controversial. Effectively, although Mazur [29] proposed that several centuries are required, of liquid nitrogen storage, for that ionizing cosmic radiation alters or damages the DNA of the cell. However, there are studies that raise a discrepancy respect that cryopre‐ servation, for several years, completely stops the processes of sperm biochemistry, but whose

Fourteen five years ago, Salisbury and Hart [16] proposed that bovine frozen sperm have a low fertility level and promote increased embryonic mortality after 1.5 years of storage at −196°C. More recently, Haugan et al. [19] based on results of field trials indicated that the

use of analysis of subjective sperm motility analysis versus our use of CASA system.

storage times do not exceed 6 years.

with the presence of sodium bicarbonate (30.9 mM) [37].

96 Cryopreservation in Eukaryotes

**Figure 3.** Representative's field captures for sperm quality parameters analyzed. (A) Plasma membrane integrity analy‐ sis, green or red fluorescents marks correspond to sperm recognized as live or dead, respectively. Images obtained with epifluorescence microscope, objective: 10×. (B) Sperm motility analysis, the tracking in red, green, blue and yel‐ low, correspond to sperm sorted according velocity: rapid, medium, slow and static, respectively. Images obtained with phase contrast microscope, objective: 10×‐negative. (C) Acrosomal integrity analysis, Coomassie blue G‐250 stained acrosome‐intact sperm or those with acrosome reacted or damaged (asterisks). Images obtained with bright field microscope, objective: 40×.


Values are expressed as mean ± SEM. Different superscript letters (a and b) indicate significant differences among storage times (one‐way ANOVA, p < 0.001).

**Table 1.** Effect of storage time at −196°C on bull sperm kinetic parameters.

Our result showed that the more important parameters of sperm quality non‐present changes associated to storage times analyzed (1–10–25–45 years). Considering that the plasma and acrosomal membrane integrity are two irreversible parameters of sperm quality, and that the motility is commonly believed to be one of the most important characteristics associated with the fertilizing ability of semen [38]. Our freezability data, analyzed as a whole, suggest that fertilizing potential of the seminal dose is commercially analyzed, independent of storage time, and it is high. In this respect, Budworth et al. [39, 40] observed significant correlation of the sperm motility and sperm velocity with the competitive fertility index. Moreover, Amann [41] reported a high level of correlation between competitive fertility index and sperm motility, VCL, VSL parameters, with 0.80, 0.68 and 0.70, respectively.

We conclude, and categorically, that the basic parameters of sperm quality of bovine semen are not affected by long‐term storage at −196°C. Complementary analysis, including other aspects as to mitochondrial metabolism, reactive oxygen species (ROS) levels, DNA fragmen‐ tation and chromatin integrity, could shed light on possible and potential changes induced for prolonged storage.

Future studies of embryo production by *in vitro* fecundation (IVF) and field trials are needed, in order to confirm effects associated to long‐term sperm storing at −196°C on fertility, embryonic viability and calving rate.
