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Cryopreservation Of Boar Semen By Vitrification

Elmer Casanova Vingua, D.V.M.,

Abstract: This study was conducted to determine the effect of introducing vitrification technology in preserving boar semen. The study consisted of three phases: Phase 1 – Effects of cryoprotectant and equilibration time on the post-thaw motility of vitrified boar semen; Phase 2 – Effect of cryoprotectant on the morphological characteristics of vitrified boar semen; and Phase 3 – Effect of cryoprotectant on the penetration capability of vitrified boar semen. Three-factor factorial design in completely randomized design (CRD) was used in Phase 1 and simple design in CRD was used in Phase 2 and Phase 3. All treatments were replicated three times. Phase 1 of the study showed that the use of ethylene glycol did not differ significantly from glycerol for the vitrification of boar semen. Seven (7) percent level of cryoprotectant agent (CPA) was significantly higher than the other treatment levels. Vitrified boar semen with 20 min equilibration time (EqT) had significantly higher mean post-thaw semen motility of 21.0 percent. Vitrified boar semen with 7 percent level of CPA and equilibrated for 20 minutes had the significant highest mean post-thaw semen motility of 36.67 percent. Phase 2 revealed that fresh semen (FS) had the highest mean percentage of normal alive sperm (75.35%) and significantly higher than the glycerol vitrified-warmed boar semen (GVWBS) with 55.72% and the ethylene glycol vitrified-warmed boar semen (EGVWBS) with 56.36%. Phase 3 showed that FS is significantly better and more capable of penetrating the oocytes in vitro with 83.1% as compared to 41.9% and 36.0% for GVWBS and EGVWBS, respectively.

Keywords: Animal physiology, Vitrification, Cryopreservation, Boar semen

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1 INTRODUCTION

Animal scientists generally agree that the most important cause of economic loss in animal industries is from reproductive inefficiency such as low conception rates and embryo mortality. Biotechnologies such as cryopreservation of semen and embryos are important and potential tools responsive to the problems of low productivity now haunting the livestock industry. These technologies are important

tools for the preservation of animal biodiversity.

Cryopreservation is a proven procedure that has been widely used for many years. Several samples of semen are collected, examined, suspended in a protective medium, and then frozen and stored at very low temperatures. Thus, preserved sperm remains viable for many years and can be used in assisted reproductive procedures. Successful cryopreservation of semen was first accomplished in 1949 by Polge and Rowson when they discovered a practical method for long-term preservation of semen of certain species by deep freezing in temperatures of -79oC by means of dry ice. In 1952, they tested the fertilizing capacity of bull sperm that had been buffered with egg yolk-sodium citrate and equilibrated with glycerol for several hours before freezing, resulting in a very satisfactory conception

rate.[1] Cryopreservation of semen, also known as freezing

or banking, allows sperms to be preserved and stored at ultra-low temperatures for future fertilization procedures. Cryopreservation period has no effect on thawed semen quality which means that freezing by immersion into liquid nitrogen does not affect the quality of semen. Semen is then preserved for an almost unlimited period of time without considerable depreciation in its quality. With this, cryopreservation of spermatozoa offers an effective solution for long-term storage of valuable genetic material and allows production of embryos with desirable genetic make-up through in vitro fertilization.[2]

It was emphasized that maximal damage to the motility apparatus, plasma membrane, and acrosomal cap of

buffalo spermatozoa occur during freezing and thawing

followed by equilibration. These damages are brought about by the formation of ice crystals during freezing.[3] To solve this problem, vitrification technique in assisted reproduction both in humans and animals was developed. It was stressed that vitrification prevents damage related to ice formation such as mechanical disruption of extracellular structures in organized tissues and organs, cellular osmotic

dehydration and shrinkage during slow freezing,

intracellular ice formation and destructive intracellular ice re-crystallization.[4] Vitrification converts a material into a glass-like solid free from ice crystals, which prevent significant damages to the egg. The 'slow freeze' method seems to increase the chance of ice crystal formation, which damages the egg.[5] In contrast to slow freezing, vitrification allows solidification of the cell(s) and the extracellular milieu into a glass-like state without the formation of ice.[6] It is a type of ultra rapid freezing method that has many advantages over conventional slow freezing methods. It is the most widely used method for embryo cryopreservation today because of the high survival rates of embryos,[7] minimal deleterious effects on post-warming embryo morphology and improves clinical outcomes,[8] and a significant improvement in embryo cryosurvival following vitrification as compared with slow-freezing.[9] Several studies on vitrification were conducted successfully in bovine embryos by open pulled straw[10], mouse embryos [11], mouse and sheep compacted morullae [12], oocytes of human and mouse by open pulled straw[13], human embryos using ethylene glycol in straws [14], mammalian oocytes and embryos[12], human oocytes and embryos,[15] human stem cells by open pulled straw [16], human spermatozoa without cryoprotectant [17], tissues and organs of rabbit [18], human sperm using open pulled straw [19], oocyte of women undergoing fertility treatment [5], on oocytes using cryotop vitrification method,[20] and on human umbilical cords. [21] In addition, vitrification successfully done in the pronuclear-stage pig embryos,[22] fish sperm and spermatogonia, [23] and grapevine. [24] Further studies revealed that a highly successful survival rates of oocytes of over 90% was observed after vitrification and warming with a fertilization rates of 75%–90%,

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Elmer Casanova Vingua, D.V.M., Ph. D.1

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pregnancy rates of 32%–65% per ET, and live-birth rates of over 50%.[25] It was also reported that vitrified embryos showed higher quality blastocysts and better pregnancy outcomes (50.4%) than slow cooling protocol (25.9%), [26]

and 89.4% survival rate on human oocytes.[27]

Furthermore, vitrification was able to freeze and bank embryos safely and reliably. It significantly enhanced the birth rate per frozen egg when combined with the use of CGH on DNA derived from the polar body (PB) of the egg.[28] On the cryoprotectant agent (CPA), it was reported that glycerol has been found to protect the sperm considerably from the freezing process.[29] It was stressed that optimal freezing rates of about 30oC/min with 3 percent glycerol for 0.5 ml straws or 50oC/min containing about 1.5 percent glycerol for 0.25 ml can maintain the motility and acrosome integrity of the boar spermatozoa.[30] It was concluded that with skim milk as extender in the cryopreservation of equine spermatozoa, ethylene glycol could serve as substitute cryoprotectant for glycerol in

stallion semen if used at the same or lower

concentrations.[31] As per observation conducted, higher

rate of recovery of motile spermatozoa after

cryopreservation when 1.0 M ethylene glycol is used rather than 1.0 M glycerol.[32] Ethylene glycol had a similar cryoprotective efficacy to glycerol that makes it a successful cryoprotectant for sperm cryopreservation in monkeys.[33]

2 OBJECTIVES OF THE STUDY

Generally, this study aimed to develop a vitrification technology for boar semen using glycerol and ethylene glycol as cryoprotectant agents. Specifically, this study aimed to (1) determine the optimum level of glycerol and ethylene glycol as cryoprotectant agents in the vitrification of boar semen; (2) determine the optimum equilibration time in the vitrification of boar semen; (3) evaluate the post-thaw morphological characteristics of vitrified-warmed boar semen; and (4) evaluate the penetrating capability of vitrified-warmed boar semen as affected by the kind and levels of cryoprotectant agent, equilibration time.

3 MATERIALS AND METHODS

Phases of the Study

There were three phases of this study. Phase 1 - Effects of cryoprotectant agent (CPA) and equilibration time on the post-thaw motility of vitrified-warmed boar semen; Phase 2 - Effect of CPA on the morphological characteristics of vitrified-warmed boar semen; and Phase 3 - Effect of CPA on the fertilizing capability vitrified-warmed boar semen.

Experimental Design

Two-factor factorial design in completely randomized design (CRD) was used in Phase 1 and simple CRD was used in the Phase 2 and Phase 3. All the treatments were replicated three times.

Methodology

Fresh boar semen (FBS) was collected and evaluated for color, volume, motility and concentration. Boar semen that passed the evaluation was used in the study. The FBS was washed using physiological saline solution and extended

agent (CPA). 50µL of extended boar semen was placed in the cryovial and immediately plunged in the liquid nitrogen. Equilibration time (EqT) was observed. After 7 days, the vitrified boar semen was thawed for 1-2 minutes at 27-32oC, placed in the tissue culture plastic dish and incubated (CO2 incubator) for 3-5 minutes at 37

o

C. Caffeine solution was added to enhance sperm motility. Vitrified-warmed boar semen (VWBS) with at least 30% motility was further

evaluated for morphology and penetration. For

morphological study, the FBS and VWBS were smeared separately in glass slide and stained with eosin-nigrosine. These were then viewed under the compound microscope. The number of alive and dead spermatozoa were counted and recorded for analysis and interpretation.For sperm penetration, the In vitro maturation and In vitro fertilization (IVM-IVF) technology were used using oocytes from slaughtered gilts/sows and spermatozoa from FBS and VWBS. Sperm motility, morphology and penetration evaluation were done using the compound microscope with TV monitor.

4 RESULTS AND DISCUSSION

In the Phase 1 of the study revealed that VWBS with glycerol (G) had a mean post-thaw motility of 14.44% while those with ethylene glycol (EG) had 14.11%. Cryoprotectant EG did not differ significantly from G for the vitrification of boar semen. On the effect of different levels of CPA on post-thaw motility, CPA at 7% level had significantly highest in mean motility with 21.11% than the other treatment levels. Vitrified boar semen equilibrated for 20 minutes had significantly highest in mean post-thaw motility with 21.0%. On the interaction effects of different levels of CPA and equilibration time (EqT), vitrified boar semen with 7% CPA and 20 minutes EqT had significantly highest in mean post-thaw motility with 36.67%. Result is presented in Table 1.

Phase 2 which was on the morphological evaluation of glycerol vitrified-warmed boar semen (GVWBS) and

Table 1. Effects of levels of cryoprotectant and equilibration time on post- thaw motility of vitrified boar semen

Level of

cryoprotectan ts (%)

Equilibration time (Minute) Levels of

cryoprotectants (Mean)

0 10 20

0 1.67f 2.5ef 8.33def 4.17c

3 10.00def 15.83bcd 15.0

0bcde 13.61

b

5 8.33def 20.00bcd 23.3

3b 17.22

ab

7 8.33def 18.33bcd 36.6

7a 21.11

a

9 9.17cdef 15.00

bcd e

21.6

7bc 15.28

b

Equilibration

Time (Mean) 7.50

c

14.33b 21.0

0a 14.28

Mean row and column of the same letter superscript are not significant at 5%

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ethylene glycol vitrified-warmed boar semen

(EGVWBScompared to fresh boar semen (FBS) revealed that FBS had significantly highest in percent mean normal alive sperm with 75.35% as compared to GVWBS with

55.72% and EGVWBS with 56.36%. Moreover, percent mean dead sperm was significantly higher in vitrified boar semen (GVWBS, 30.61% and EGVWBS, 27.49%) than FBS with 8.25%. Result is presented in Table 2.

Phase 3 of the study revealed that FBS registered the highest penetrated oocytes of 83.17% (monospermy, 46.67% and polyspermy, 36.5%), while GVWBS had 41.9% (monospermy, 23.8% and polyspermy, 18.1%), EGVWBS

had 36.0% (monospermy, 19.6% and polyspermy, 16.4%). The result revealed that FBS was significantly better and more capable of penetrating/ fertilizing the oocytes in vitro than VWBS.

5 CONCLUSION

Based on the findings of the study, the researcher concluded that:

1. Glycerol and ethylene glycol were found to be

effective CPA for the vitrification of boar semen;

2. Vitrified boar semen with 7% level of CPA was

significantly highest in mean post-thaw motility; 3. Vitrified boar semen equilibrated at 20 min had

significantly highest in mean post-thaw motility; 4. Vitrification of boar semen at 7% level of CPA and

20 min EqT had significantly highest in mean post-thaw motility;

5. Vitrified boar semen had significantly lesser live normal sperm compared to FBS;

6. Vitrified boar semen was significantly less capable of penetrating the porcine oocytes in vitro compared to FBS.

6 RECOMMENDATION

Based on the result of the study, the following are hereby recommended:

1. Glycerol is the universally accepted cryoprotectant agent for vitrification technology; however, in its absence, ethylene glycol can be used which is similarly effective and efficient.

2. When vitrifying boar semen, the level of

cryoprotectant should be at 7 percent level and 20 minutes equilibration time.

3. Because of its potential as a ready source of semen for artificial insemination in swine, a similar

Table 2. Percent mean morphological characteristics of fresh and vitrified-warmed boar semen Nature of semen Morphological characteristics of boar sperm (%)

Normal Abnormal Dead

FBS 75.35a 16.73 8.25b

GVWBS 55.72b 13.67 30.61a

EGVWBS 56.36b 16.15 27.49a

P-value 0.001472 2.291 0.002953

Mean columns of the same letter superscript are not significant at 5% significant (P<0.05) not significant (P>0.05)

FBS- Fresh boar semen

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study is strongly recommended focusing on the volume and thawing procedures.

7

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Figure

Table 1. Effects of levels of cryoprotectant and equilibration time on post- thaw motility of vitrified boar semen
Table 2Nature of semen . Percent mean morphological characteristics of fresh and vitrified-warmed boar semen  Morphological characteristics of boar sperm (%)

References

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