**2. Materials and methodology**

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 concep‐ tion 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 [8–10]. Meanwhile, studies of frozen

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

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

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

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

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

packaging technique, some factors other than storage are responsible.

semen stored at −196°C have shown a consistently high non‐return rate [11–14].

delayed return [9, 15].

92 Cryopreservation in Eukaryotes

banks [20, 21].

normal fertility rate was evidenced.

#### **2.1. Seminal doses, race of donor and processing**

In this study, a total of 75 commercial doses from bulls Friesian breed were used. The cryo‐ preserved 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), belonAus‐ tral 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 Thermos‐ stated 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.

**Figure 1.** Different freezing packaging system used in this study.

#### **2.2. Sperm quality analysis of seminal doses**

#### *2.2.1. 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.

#### *2.2.2. Sperm motility assessment*

Sperm motility was assessed using the CASA system (Motility module of the Sperm Class Analyser®, Microptic, Spain), according to Ramírez et al. [35], with modifications. A total of 6 μL aliquots of samples was then placed on a prewarmed (37°C) slide and covered with a 24 mm2 coverslip. The motility analysis by CASA system was based on the analysis of 25 consecutive, digitalized photographic images taken over a time lapse of 1 s, obtained from a single field using a negative objective (10× magnification) and a phase contrast microscope (Nikon E200, Japan), coupled to a high‐velocity camera (Basler AG, Germany, scA780 54tc). Four or seven separate fields were taken for each sample (at less 500 spermatozoa analyzed). Sperm motility parameters were as follows: curvilinear velocity (VCL); linear velocity (VSL); mean velocity (VAP); linearity coefficient (LIN): (VSL/VCL) × 100(%). Straightness coefficient (STR): (VSL/VAP) × 100(%). Wobble coefficient (WOB): (VAP/VCL) × 100(%). Mean amplitude of lateral head displacement (ALH); frequency of head displacement (BCF). Bovine configu‐ ration of CASA system used was as follows—capture: 25 frames/s; particle area range: 5–70 μm2 ; classification according to velocity (VAP): static < 10 μm/s < slow < 25 μm/s < medium < 50 μm/s < rapid. The progressive motility was defined as the percentage of spermatozoa showing an STR above 70%.

#### *2.2.3. 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 permea‐ bilized 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.

#### **2.3. Statistical analysis**

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 Thermos‐ stated water bath at 40°C for 55 s; short straw (Mini‐Tubes) and fine straw samples were thawed

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

Sperm motility was assessed using the CASA system (Motility module of the Sperm Class Analyser®, Microptic, Spain), according to Ramírez et al. [35], with modifications. A total of 6 μL aliquots of samples was then placed on a prewarmed (37°C) slide and covered with a 24

 coverslip. The motility analysis by CASA system was based on the analysis of 25 consecutive, digitalized photographic images taken over a time lapse of 1 s, obtained from a single field using a negative objective (10× magnification) and a phase contrast microscope (Nikon E200, Japan), coupled to a high‐velocity camera (Basler AG, Germany, scA780 54tc). Four or seven separate fields were taken for each sample (at less 500 spermatozoa analyzed). Sperm motility parameters were as follows: curvilinear velocity (VCL); linear velocity (VSL); mean velocity (VAP); linearity coefficient (LIN): (VSL/VCL) × 100(%). Straightness coefficient

in a thermo‐stated water bath at 37°C for 30 s.

94 Cryopreservation in Eukaryotes

**Figure 1.** Different freezing packaging system used in this study.

*2.2.1. Plasma membrane integrity (viability assessment)*

**2.2. Sperm quality analysis of seminal doses**

each sample.

mm2

*2.2.2. Sperm motility assessment*

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.
