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Brazilian Archives of Biology and Technology

Print version ISSN 1516-8913

Braz. arch. biol. technol. vol.57 no.2 Curitiba Mar./Apr. 2014 



Development of single tubing-type canister for cryo-storage of bull semen and their effect on sperm motility and viability



Mohd Iswadi IsmailI, III, *; Khairul OsmanII; Siti Fatimah IbrahimI; Farah Hanan Fatihah JaafarI; Nur Azianie Abd GhaniI; Fazly Ann ZainalabidinIII; Abas Mazni OthmanIV

IDepartment of Physiology; The National University of Malaysia; Kuala Lumpur - Malaysia
IIForensic Science Programme; Faculty of Health Sciences; The National University of Malaysia; Kuala Lumpur - Malaysia
IIIVeterinary Research Institute; Department of Veterinary Services; Ipoh, Perak - Malaysia
IVMalaysian Agricultural Research and Development Institute; 43400 Serdang - Selangor - Malaysia




The objective of this study was to evaluate the potential of using single tubing-type canister on sperm quality. Semen was collected from the Bali cattle bull by electroejaculation technique and was cryopreserved in liquid nitrogen using slow freezing cryopreservation method. Two type of canister volume was used in this study; commercial canister (342.25π x 278 mm2) and single tubing-type canister (4π x 90 mm2). Makler counting chamber and computer assisted sperm analyzer (CASA) were used to evaluate the sperm motility and viability of post-thaw sperm. Results showed that the bull sperm motility and viability at the bottom of tubing-type canister was statistically higher and significant as compared to the commercial canister (p<0.05). Significant changes were found in sperm kinetics (VCL, VAP, VSL) of tubing-type canister compared to commercial canister. No significant changes in the motility and viability of the bull sperm at the top of tubing-type canister and commercial canister. There were no significant changes in sperm progression (LIN, WOB, PROG) in both the canisters. Developed tubing-type canister in this study showed potential as an alternative to be used in bull sperm cryo-storage.

Keywords: Cryopreservation, sperm, canister, motility, viability.




There has been considerable attention on the determination of optimum conditions for cryopreservation process and cryo-storage of semen of domestic species (Curry 2000; Barbas and Mascarenhas 2009). Many benefits have resulted from the process of cryopreservation. The most important benefit of all is the ability to store the sperm for long periods of time and still be able to use a portion of it later. Furthermore, semen cryopreservation offers many advantages to the livestock industry and to the maintaining sperm storage of the threatened and endangered wild species (Fickel et al. 2007). Cryopreservation is a technique that involves a very wide range of temperature changes. However, the biggest obstacle to exploiting cryopreserved semen of many species is that cooling, freezing and thawing, which generally damage sperm membrane structures, causes fewer viable and motile sperm (Hammerstedt et al. 1990).

There are very limited reports on the effect of various canisters dimension on the sperm during the semen cryo-storage and canister volume of 342.25π x 278 mm2 is commonly used for bull semen cryo-storage. There are various types of considerable materials for bull semen cryo-storage (e.g., vials, straws, etc). Routinely, either 0.25 mL or 0.5 mL straws have been a suitable choice for bull semen cryo-storage (Sansone et al. 2000; Vishwanath and Shannon 2000). In this study, a tubing-type canister was developed with the hypothesis that the sperm motility and viability could be related to the use of suitable canister dimension in cryo-storage.



Semen collection

Fresh semen samples were collected from the Bali cattle bulls using electroejaculation technique by a trained veterinarian. Makler counting chamber (Sefi-Medical Instruments Ltd) was used to determine the sperm concentration. All the samples were added to a Bioxcell® medium (IMV Technologies, France) before the cryopresevation process. Motility and viability of the sperm were determined in post-thawed sample.

Cryopreservation technique

In this study, slow freezing technique was used. After adding with Bioxcell® medium, the samples were then put into a 4°C cooling cabinet for 4 h, and then sucked into the 0.25 mL cryo-straw using the vacuum pump attached with filling nozzle. For each straw, the final concentration of the sperm was set at 25 x 106 sperm/mL. After keeping the straws above the liquid nitrogen vapor (-60 to -120°C) for nine minutes, they were then plunged and stored in liquid nitrogen (-196°C) for 24 h before the motility and viability assessment.

Semen storage

The straws were stored in two types of canister; commercial canister (342.25π x 278 mm2) and custom-made tubing-type canister (4π x 90 mm2) (Fig. 1). The depth of liquid nitrogen tank used was between 320 to 350 mm. The level of the liquid nitrogen on the cylinder was fill and maintained as minimum as 50 mm above the top of the canister during the storage period. A bundle of straws was stored in commercial canister and one straw per tubing-type canister.



Total of twenty-four 0.25 mL straws were used; eight straws in the commercial canister, eight in upper tubing-type canister and eight in the bottom of tubing-type canister.

Sperm motility assessment

Sperm motility was graded from a to d, according to the World Health Organization (WHO 1999) Manual criteria as follows: a for fast progressive motility; b for slow progressive motility; c for non-progressive sperm; d for immotile sperm. A part of the semen was analyzed by computer assisted sperm analysis (CASA) for sperm velocity and progression assessment.

Statistical analysis

Data were analyzed using SPSS unpaired t-test at significant level, p<0.05. All the values are shown as means ± SEM.



A custom-made tubing-type canister was designed, which fitted enough the 0.25 mL cryo-straw for each tube. This canister was 4.0 mm in diameter and 330 mm height with two 90 mm length of tubes on each cane (top and bottom tube). On the other hand, commercial canister was 37 mm in diameter and 515 mm height with 278 mm length of bucket. The volume of tubing-type canister was 4π x 90 mm2, which was less when compared to the volume of commercial canister (342.25π x 278 mm2).

Fast progressive, slow progressive, non-progressive and immotile sperm was higher in the commercial canister as compared to the tubing-type canister (Fig. 2). Results in this study showed that post-thawed sperm motility were 21.50±3.32% (commercial canister), 12.41±2.86% (top tubing-type canister) and 7.63±3.75% (bottom tubing-type canister). The sperm viability was 28.00±6.27% (commercial canister), 26.14±5.36% (top tubing-type canister) and 15.75±4.69% (bottom tubing-type canister). However, there were no significant changes in all the motility class (a-d) and viability between the commercial and tubing-type (top and bottom) canister (Fig. 3).





Results by CASA analysis showed that the sperm velocity in the commercial canister were 598.29±51.4 µm/sec (VCL), 326.03±30.2 µm/sec (VAP) and 260.39±24.52 µm/sec (VSL), which in the top tubing-type canister were 850.85±62.64 µm/sec (VCL), 473.66±40.69 µm/sec (VAP), 373.19±33.10 µm/sec (VSL) and in bottom tubing-type canister were 625.08±42.78 µm/sec (VCL), 359.34±21.04 µm/sec (VAP) and 283.57±17.14 µm/sec (VSL). Significantly higher (p<0.05) sperm velocity (VCL, VAP, VSL) was recorded in the top tubing-type canister compared to the commercial and bottom of tubing-type canister (Fig. 4). Results of sperm progression in the commercial canister were 0.82±0.01% (LIN), 0.56±0.02% (WOB) and 4.09±0.12% (PROG), which in the top tubing-type canister were 0.79±0.01% (LIN), 0.57±0.02% (WOB), 4.04±0.12% (PROG) and in bottom tubing-type canister were 0.80±0.01% (LIN), 0.58±0.01% (WOB) and 3.82±0.08% (PROG). There were no significant changes in sperm progression (LIN, WOB, PROG) between the commercial and tubing-type (top and bottom) canister (Fig. 5).





The temperature of storage is an important consideration for cryo-storage the semen. Gandini et al. (2007) reported freezing and cryo-storage of semen as being developed for most livestock species. The use of freezing and cryopreservation techniques for semen storage is tremendously improved, mostly with an aim for genetic conservation of livestock breed population. Various techniques and processes have been used for semen storage in long period of time such as storage at 5°C (Bartlett and Van Demark 1962), storage in deep freezer (-40 and -80°C) (Polge and Rowson 1952), and cryo-storage at ultra-low temperature (-196°C) (Fickel et al. 2007). The efficiency and utilization of stored semen could be significantly increased if the effects of freezing and cryopreservation can be overcome. On the other hand, with more advanced cryogenic vessels, it was found possible to maintain the sperm in a frozen state for a very long period. The discovery of glycerol as an effective cryoprotective agent introduced a completely new system of semen storage and a method commonly used following the example set by Polge et al. (1949). Since then, various commercial cryoprotective agents such as Triladyl®, Biladyl®, Bioxcell® and Andromed® have been developed and used for bull semen cryopreservation (Janett et al. 2005; Muino et al. 2007). Bioxcell® was widely used and reported suitable in cryopreservation of domestic bulls and was attributed to the higher concentrations of glutathione, which protected the sperm against oxidative stress and reactive oxygen species (Stradaioli et al. 2007). The level of glutathione in Bioxcell® was reported sufficient enough to protect the sperm from oxidative stress during freeze-thawing process. Bioxcell® medium also provides nutrients and contains cryoprotectant agent to extend the longevity of the sperm during the cryopreservation process. Based on study by Stradaioli et al. (2007), Bioxcell® was selected and used as cryprotective agent in this study.

Eriksson (2000) reported the technique of freezing semen in 0.25 or 0.5 mL straws in liquid nitrogen, which has been universally used. This technology is well-established and all the equipment required for filling the semen in straws and racking them up for freezing are readily available. Although there have been considerable studies in determining the optimum cooling rates and adapting the cooling curves for individual bulls, there has been very little progress in the practical sense. Nowadays, semen storage organisational continues to use commercially available canister where the straws containing semen are subjected to cryo-stored in liquid nitrogen. The advantage of the commercial canister is that all the straws in any given freezing cycle are subjected to cryopreserve in large quantity of straws, because the straws are placed in one big bucket of canister. However, the repeatability of movement of the whole straws in the canister to pick up the wanted straw for thawing process affects the sperm due to the changes of the temperature. As an alternative, the development of single-tubing type canister could minimise or prevent the effect of the temperature with only the wanted straw being picked up without disrupting other straws.

Foote (1984) reported the optimal conditions for freezing of bull semen, which led to considerable improvement of sperm survival after freezing and thawing. The results of this study revealed the motility and viability of freezing and thawing sperm in single-tubing type canister as lower compared to the sperm in commercial canister. Results by CASA showed that the sperm kinetics (VCL, VAP and VSL) was significantly higher than the sperm stored in commercial canister. There were statistically no significant changes of sperm progression (LIN, WOB and PROG) in both the canisters; single-tubing type and commercial canister. Result of this study was supported by the earlier findings of Osman et al. (2008), which reported the sperm kinetics highly associated with the DNA damage of the sperm. Using CASA results, they found that VSL, WOB and VCL were important factors in determining the overall condition of a particular sperm with 83.6% accuracy using the correlation and regression (C&R) statistical analysis model. Combination assessment of VSL, WOB and VCL could be used in determining the DNA integrity of the freeze-thawed sperm.

On the other hand, many studies have reported the physical changes of the sperm associated with freezing and thawing process (Bag et al. 2002; Maldijan et al. 2005; Chaveiro et al. 2006; Leahy and Gadella 2011). The physical changes during the process of freezing and thawing are quite dramatic (Saacke 1982). These were due to the external environment of the semen in the straws during cryo-storage in the canister and the process of freezing and thawing. The physical effects of cooling and freezing on the sperm result in a number of changes in the external environment in terms of water and solute movement. The rate at which water moves out of the cell during this process plays an important role in determining the cooling rates, which has to be optimised for cell survival after thawing (Mazur 1977). The effect of freezing on the sperm was due to the stresses associated with freezing, which was primarily due to the changes in the temperature that spermatozoa were subjected during the process of cooling. The injurious effects of the media components and cryoprotectants agent themselves during the process and finally the effects of thawing were other factors.

The general theory is that the rapid thawing of semen is advantageous to prevent the injury during rewarming. Mazur (1984) observed that rapid thawing prevented the possibility of re-crystallisation of water molecules, which could be injurious to cell membranes. A potential problem in slow thawing is the osmotic change due to ingress of water during the process, as this is considered to be more damaging than water egress during the freezing (Curry and Watson 1994). Practical considerations regarding the temperature at which semen used in field conditions should be thawed to obtain maximum fertility.



Based on the results of this study, it could be concluded that tubing-type canister showed potential compared to the commercial canister and alternatively available to be used in bull sperm cryo-storage. The future direction of research on freezing and cryo-storage semen should be directed towards improvement of freezing protocols to allow a lower number of sperm to be included per straw and tubing unit. For any substantial gain to be made in this direction, future work also needs to concentrate on the diluents composition, repeatable cooling rates to protect the sperm cells during the freezing process and the effect of freezing and thawing at microenvironment of the sperm.



We would like to extend our gratitude to the Ministry of Science, Technology and Innovation (MOSTI) for providing research grant (FF 162-2009) to support this study.



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Received: January 24, 2013
Accepted: December 23, 2013



* Author for correspondence:

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