Long‐Term Storing of Frozen Semen at −196°C does not Affect the Post-Thaw Sperm Quality of Bull Semen Long ‐ Term Storing of Frozen Semen at −196°C does not Affect the Post-Thaw Sperm Quality of Bull Semen

Today, it is theoretically assumed that frozen storage of semen doses in liquid nitrogen guarantees sperm functionality indefinitely. However, there are few studies that objectively evaluate the effects of long‐term storage on sperm quality parameters. In this study, we show a freezability analysis of bull semen stored for 1, 10, 25, 40 and 45 years at −196°C. Sperm viability and full sperm motility were analyzed by CASA system, and acrosome integrity was assessed with Coomassie blue staining. Our results showed that sperm viability and total sperm motility were not affected by long‐term cryopreserva‐ tion at −196°C. Specifically, we did not find any significant differences (p > 0.05) associated between different long‐time storing analyzed; both parameters showed optimal values of sperm viability and total sperm motility (both over 60%). Additionally, the acrosomal integrity parameter was not affected, showing an optimal range (87±1.6 ‐ 95±0.5%). We conclude that the sperm quality of bovine semen is not affected by long‐ term storage at −196°C. However, future field trials will be necessary in order to validate that both fertility and embryo viability are maintained for the times analyzed.


Introduction
Cryopreservation protocols for the bull used in the animal production industry began in the 1950s [1]. Since then, both the packaging type and cryopreservation system were changing on the time. Primary considerations in the selection of a system for packaging semen were fertility, insemination preference, ease of handling, ease of identification, freedom from contamination, economics of storage and efficiency of ejaculates [2]. Sperm are commonly packaged in one of three ways: (a) glass ampoules, normally containing 0.5-1.2 ml of frozen semen; (b) pellets containing about 0.1 ml; and (c) polyvinyl chloride straws with a volume of 0.25-0.5 ml.
Early field trials showed that the bovine semen frozen to −79°C and packed on dry ice could still yield high fertility [3]. Regarding to time storage factor, studies of sperm motility have indicated a descent in sperm viability after storage [4,5]. On the other hand, field trials carried out at the Reading Cattle Breeding Centre (Great Britain, 1960) indicated no effect on conception rate when using long-term semen stored in a dry ice alcohol mixture for 4 years [6].
Until the 1970s, it was thought that frozen semen could be indistinctly stored in mechanical freezers at about −25°C, in solid carbon dioxide at −79°C, or in liquid nitrogen at −196°C. However, an inverse relationship between preservation of sperm viability and storage temperature was shown [7]. Briefly, most frozen semen was stored in a mixture of dry ice and alcohol at −79°C, which ultimately decreased fertility [8][9][10]. Meanwhile, studies of frozen semen stored at −196°C have shown a consistently high non-return rate [11][12][13][14].
Furthermore, since the 1970s, there have been mentions that deterioration continues even when sperm are stored in liquid nitrogen; suspecting that aging of spermatozoa may occur if semen is stored for long periods of time, and this may be associated with embryo mortality and delayed return [9,15].
Studies by Salisbury and Hart [16] suggested that bovine frozen sperm have a low fertility level and promote increased embryonic mortality after 18 months of storage at −196°C, but other studies have been unable to confirm this. In this context, Strom [14] found no evidence of reduced fertility when approximately 60,000 inseminations were performed with semen packaged in pellets, following storage in liquid nitrogen at −196°C for approximately 1-1.5 years. Cassou [17] reported no difference in fertility after 285,551 inseminations with frozen semen in straws were stored at −196°C for up to 4.5 years. Similarly, a field trials of Roettger et al [18], with 100,000 inseminations and using frozen semen stored at −196°C for 5 years, a normal fertility rate was evidenced.
Field trials using frozen semen packaged in ampoules, pellets and straws have indicated that the influence of packaging methods in fertility has been inconsistent [2]. Therefore, according to these authors, if the semen fertility stored in LN is reduced with time, regardless of packaging technique, some factors other than storage are responsible.
Today, cryopreservation in liquid nitrogen (−196°C) is a technique that allows for long-term storage of spermatozoa [19]. This is a highly practical method in breeding programs for domestic animals and is used to maintain the establishment and genetic diversity of gene banks [20,21].
Cryopreservation requires many stages during cooling/freezing and thawing procedures, which interactively affect its success [22,23]. On the other hand, it is assumed that storage period in deep freezing does not affect sperm viability [24,25], and there is argument that spermatozoa retain their fertilizing potential indefinitely when stored at −79°C in dry ice, or at −196°C in liquid nitrogen [26]. However, there is a scarcity of studies designed in order to detect a decrease in reproductive performance of cryopreserved semen as a function of storage time.
In this respect, Mazur [27] proposed that, by accumulative cosmic radiation, more than 3,000 years. Then the question arises, how long cells can be stored in liquid nitrogen without suffering damage? This question is probably irrelevant if the cellular storage temperature is below −120°C, where chemical reactions do not occur in a human timescale. Moreover, at −196°C, the thermally driven reactions only can occur on a geological timescale [28].
There is the possibility of slow accumulation of direct damage from ionizing radiation, but this becomes significant only after centuries of storage [29]. Yet, as previously mentioned, insemination trials with frozen stored semen [16] suggest a far shorter time period of optimal semen storage at the above-mentioned low temperatures, in consideration of fertility rate maintenance. However, other evidence suggests that this could be due to inadequate maintenance of temperatures [26]. There are two studies that strongly reinforce the idea that fertilization potential is maintained in long-term storage in liquid nitrogen. Specifically, the in vitro fertilization (FIV) was obtained using frozen spermatozoa, stored in nitrogen liquid during 37 and 27 years, for bovine and human, respectively [30,31].
In relation to sperm quality parameters, there are few studies that objectively evaluate the effects of long-term sperm storage. Leibo et al. [30] reported a normal bovine sperm motility after 37 years, and Rofeim and Gilbert [32] reported no statistical reduction in human sperm quality after 5 years of follow-up. More recently, although it was not analyzed by computerassisted sperm analysis (CASA system), Malik et al. [33] showed that the viability and motility of thawed sperm stored in liquid nitrogen during 6 years were lower than 1-to 2-year storage .
The main goal of this study was to assess, through CASA system, the main sperm quality parameters of cryopreserved and stored bull semen in liquid nitrogen for 10, 25, 40 and 45 years.

Seminal doses, race of donor and processing
In this study, a total of 75 commercial doses from bulls Friesian breed were used. The cryopreserved germplasm were defined and divided into five groups according to the storage time. For each group, 15 seminal doses from five different donors (three each) were considered. All seminal doses used were collected, processed, packaged, cryopreserved and stored (−196°C) using commercial standard procedures by Center of Artificial Insemination (CIA), belonAustral University Chile (UACh).
The mean storage times or groups of the semen doses analyzed were the following: 45, 40, 25 and 10 years, which were packaged in glass ampoules, pellets, short straws and fine straws, respectively. As a control, commercial frozen doses cryopreserved in fine straws and stored in liquid nitrogen for 1 year were used. The following thawing protocols were used, in accordance with cryopreservation packaging supports (that is showed in Figure 1): Ampoule samples were thawed in thermo-stated water bath at 50°C for 75 s; pellet samples were thawed in a Thermosstated water bath at 40°C for 55 s; short straw (Mini-Tubes) and fine straw samples were thawed in a thermo-stated water bath at 37°C for 30 s.

Plasma membrane integrity (viability assessment)
Plasma membrane integrity was determined using the acridine orange (AO)/propidium iodide (PI) double-staining technique, according to Córdova et al. [34], with modifications. Briefly, post-thawing samples (3 μL) were mixed (1:1) with a staining aqueous solution composed of 20 μM AO and 10 μM PI in a tempered microscope slide. Stained samples were analyzed using the CASA System (viability module of the Sperm Class Analyser ® , Microptic, Spain) coupled to an epifluorescence microscope (Nikon E200, Japan) with a high-velocity camera (Basler AG, Germany). Viability percentages were established from a minimum of 1000 spermatozoa for each sample.

Sperm acrosomal integrity assessment
The structural status of sperm acrosomes was assessed using Coomassie G-250 staining, according to Larson et al. [36]. Briefly, sperm aliquots were washed in TBS, fixed and permeabilized for at least 30 min at 4°C in 100% methanol. Permeabilized spermatozoa dried onto slides were then covered with a droplet of staining solution (0.22% W/V Coomassie blue G-250; 50% methanol and 10% glacial acetic). The samples were washed with excess of bidistilled water, dried and observed under 100 × oil immersion lens. Percentage of stained cells was determined by counting of at less 300 spermatozoa.

Statistical analysis
Statistical analyses were performed using one-way ANOVA with post hoc Bonferroni multiple comparison tests. For the analysis, we used GraphPAD (Prism 6) software and differences were considered significant and highly significant for p values of <0.05 and <0.01, respectively.

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 associated 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  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 cryopreservation, 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.  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 fragmentation 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.