**1. 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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

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.Thenthe questionarises,how long cells canbe storedinliquidnitrogen without suffering damage?Thisquestionisprobablyirrelevantifthecellular storagetemperatureisbelow−120°C, where chemicalreactionsdonot occurinahumantimescale.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, insemi‐ nation 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 mainte‐ nance. 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 computer‐ assisted 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.
