Cryopreservation of mouse morulae in glycerol, sucrose and honeybee royal jelly

Criopreservação de mórulas de camundongos em glicerol, sacarose e geléia real

Cryopreservation of mouse morulae in glycerol, sucrose and honeybee royal jelly^*

Criopreservação de mórulas de camundongos em glicerol, sacarose e geléia real

José Antônio VISINTIN¹; José Fernando GARCIA¹; Thais PANTANO¹; Mayra Elena Ortiz D’ÁVILA ASSUMPÇÃO¹

CORRESPONDENCE TO:

José Antônio Visintin

Departamento de Reprodução Animal

Faculdade de Medicina Veterinária e Zootecnia da USP

Cidade Universitária Armando de Salles Oliveira

Av. Prof. Orlando Marques de Paiva, 87

05508-000 – São Paulo – SP

e-mail – [e-mail]

SUMMARY

Compacted mouse morulae were frozen at 0.3ºC/min. or 0.5ºC/min. from –6ºC to –24ºC or –32ºC in 10% of glycerol plus different sucrose concentrations with or without 0.1% of honeybee royal jelly. Embryos were thawed in water bath at 22ºC for 20 seconds and cryoprotectant dilution was done in three steps. Embryos were cultured in Whitten’s medium for 24, 48 and 72 hours at 37ºC, 5% of CO₂ and 100% of humidity. The in vitro development ranged from 56.6% to 100% after 72 hours. Expanded blastocysts were transferred to pseudopregnant recipients on the third day of the estrous cycle. Viable fetuses rates for embryos frozen to –24 or –32ºC at 0.3ºC/minute in 10% glycerol + 10% sucrose, 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly, 10% glycerol + 0.1% honeybee royal jelly or 10% glycerol were respectively: 28.1% and 13.6%, 48.7% and 31.9%, 28.6% and 13.2%, 20.0% and 42.4%. Viable fetuses for embryos frozen to –24ºC or –32ºC at 0.5ºC/minute in 10% glycerol + 10% sucrose or 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly were respectively 29.0% and 15.3%, 48.8% and 32.0%. We can conclude that addition of 10% sucrose to 10% glycerol was efficient for embryo freezing at 0.3 or 0.5ºC/minute and plunged in liquid nitrogen at –24ºC. The honeybee royal jelly addition provided higher viable fetuses rates when embryos were cooled at 0.3 or 0.5ºC/minute and plunged in liquid nitrogen at –24ºC.

UNITERMS: Mice; Embryo; Cryopreservation; Sucrose; Royal jelly.

INTRODUCTION

The embryo freezing techniques in liquid nitrogen allowed the widespread practice of embryo transferring and trading between distant regions, diminishing the cost and sanitary risk⁵.

Jackowski et al.⁶ noted that a high survival rate for most mammalian cells frozen at below zero temperatures needs the presence of cryoprotectants and that the influx kinetics of these substances can be influenced by the stage of embryo development and the temperature¹⁰. The cryoprotectant decreases the damage of the solution effect at low temperatures, by diminishing the intracellular solutes and cell dehydration.

Many factors must be analyzed when the embryos are in different cryoprotectant solutions before freezing, like the time and temperature of equilibrium, the toxicity and concentration of the cryoprotectant and the stage of embryo development⁸.

Bürkle et al.³ studied the effect of glycerol associated with honeybee royal jelly in freezing mice embryos. They verified that embryonic development, implantation and fetuses rates larger in the group with 0.1% of honeybee royal jelly and 10% of glycerol, due to cellular membrane stabilization.

Visintin et al.¹³ froze mouse morulae in four solutions containing glycerol plus sucrose and honeybee royal jelly. They verified that the addition of 0.1% of honeybee royal jelly in the cryoprotectant solution containing 10% of glycerol or 10% of glycerol plus 10% of sucrose resulted in better blastocysts and fetuses rates. But no potential effect of the honeybee royal jelly was noted on the freezing solution containing 10% of glycerol and 10% of sucrose.

Dobrinski⁴ observed that pregnancy rates of frozen embryos are not equivalent with non frozen embryos. This difference is related to the destruction of embryonary cell architecture and its cytoskeleton during the freezing process, due to intracellular ice formation, damaging the plasmatic membrane.

According with Leibo⁷, embryos have high concentration of cryoprotectant during thawing, which must be removed to prevent the excessive water influx that causes osmotic shock.

Then this removal may be done stepwise with decreasing cryoprotectant concentration or addition of sucrose in the medium, which decreases the number of steps⁹, increasing post-thawing embryonic survival rates.

The sucrose allows good diffusion of the cryoprotectant and controls the increase of cellular volume during cryoprotectant removal, keeping the equilibrium of the initial extracellular osmolarity¹².

The association between glycerol and sucrose was employed by Williams; Johnson¹⁴, which indicated that 2.0M of glycerol plus 0.5M of sucrose presented 84% of embryo development.

The objective of this research was to find an association of different cryoprotectants that allows the increase of freezing speeds, achieving high pregnancy rates and simplifying embryo transfer technology in the field.

MATERIAL AND METHOD

Cross-bred mice from CB6F1/Han lineage were superovulated with 5IU of PMSG (15PM) and 5IU of hCG 46 hours later (13PM). The mice were matted just after hCG injection and observed the vaginal plug in the following day (D.1) at 7AM.

On the third day (D.3) embryos were recovered and classified based in Agrawala¹.

In the Experiment I, to test the in vitro development, the excellent compacted morulae were frozen in 8 different groups at 0,3ºC/minute from –6ºC to –24ºC (I = 10% glycerol; II = 10% glycerol + 0.1% honeybee royal jelly; III = 10% glycerol + 2% sucrose; IV = 10% glycerol + 4% sucrose; V = 10% glycerol + 6% sucrose; VI = 10% glycerol + 8% sucrose; VII = 10% glycerol + 10% sucrose and VIII = Control). In the Experiment II, to test the in vivo development, the embryos were frozen in different groups: 3 at 0,3ºC/minute and 3 at 0,5ºC/minute from –6ºC to –24ºC or 3 at 0,3ºC/minute and 3 at 0,5ºC/minute from –6ºC to –32ºC (XI = 10% glycerol + 10% sucrose; XII = 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly; XIII = 10% glycerol + 0.1% honeybee royal jelly; XIV = 10% glycerol; XV = 10% glycerol + 10% sucrose and XVI = 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly).

For freezing, embryos were held in medium from 10-15 minutes, loaded in straws, plunged in alcohol at –6ºC and kept in this temperature for 5 minutes for equilibrium. After seeding, temperature was decreased at 0.3ºC/minute or 0.5ºC/minute from –6ºC to –24ºC or –32ºC, when straws where plunged in liquid nitrogen.

The embryos were thawed in water bath (22ºC) for 20 seconds and the cryoprotectants removed in three decreasing glycerol concentration solutions (6.6%; 3.3% and 0.0%) with sucrose (10%).

The embryos were cultured in Whitten medium at 37ºC, 5% of CO₂ and 100% of humidity for 24, 48 and 72 hours (Experiment I) and for 24 (Experiment II).

The in vitro development was considered when the embryos arrived the early blastocyst, blastocyst, expanded blastocyst and hatching blastocyst stages (Experiment I).

Expanded blastocysts were transferred to recipients to evaluate implantation, resorption, and fetuses rates (Experiment II). The recipients were sacrificed at 11 days after embryos transfer.

The statistical analysis was done by Chi-square² when comparing two different treatments and analysis of variance (ANOVA) and Tukey’s test¹¹ when comparing three or more treatments (a = 0.05).

RESULTS

The results of morphological evaluation of compacted morulae frozen at 0.3ºC/minute from –6ºC to –24ºC in different cryoprotectant solutions and cultured in vitro for 24, 48 and 72 hours can be observed in Tab. 1.

Table 1

Development of embryos frozen at 0.3ºC/minute from –6ºC to –24ºC in different cryoprotectant solutions after 24, 48 and 72 hours in culture, São Paulo, 1999.

I – 10% glycerol; II – 10% glycerol + 0.1% honeybee royal jelly; III – 10% glycerol + 2% sucrose; IV – 10% glycerol + 4% sucrose; V – 10% glycerol + 6% sucrose; VI – 10% glycerol + 8% sucrose; VII – 10% glycerol + 10% sucrose; VIII – Control. Different letters in the same column means statistical difference (a= 0.05).

Tab. 2 and 3 show implantation, resorption and fetuses rates of embryos frozen at 0.3ºC/minute and 0.5ºC/minute from –6ºC to –24ºC in different cryoprotectant solutions, thawed and cultured in vitro during 24 hours.

Table 2

Implantation, Resorption and fetuses rates of embryos frozen at 0.3ºC/minute from –6ºC to –24ºC, São Paulo, 1999.

XI – 10% glycerol + 10% sucrose; XII – 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly; XIII – 10% glycerol + 0.1% honeybee royal jelly; XIV – 10% glycerol. Different letters in the same column means statistical difference (a = 0.05).

Table 3

Implantation, Resorption and fetuses rates of embryos frozen at 0.5ºC/minute from –6ºC to –24ºC, São Paulo, 1999.

XV – 10% glycerol + 10% sucrose; XVI – 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly. Different letters in the same column means statistical difference (a = 0.05).

Tab. 4 and 5 show implantation, resorption and fetuses rates of embryos frozen at 0.3ºC/minute and 0.5ºC/minute from –6ºC to –32ºC in different cryoprotectant solutions, thawed and cultured in vitro during 24 hours.

Table 4

Implantation, Resorption and fetuses rates of embryos frozen at 0.3ºC/minute from –6ºC to –32ºC, São Paulo, 1999.

XI – 10% glycerol + 10% sucrose honeybee royal jelly; XIII – 10% glycerol + 0.1% honeybee royal jelly; XIV – 10% glycerol. Different letters in the same column means statistical difference (a = 0.05).

Table 5

Implantation, Resorption and fetuses rates of embryos frozen at 0.5ºC/minute from –6ºC to –32ºC, São Paulo, 1999.

XV – 10% glycerol + 10% sucrose; XVI – 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly. Different letters in the same column means statistical difference (a = 0.05).

DISCUSSION

The permeability to the cryoprotectant influences the cellular response to the freezing and thawing process. This happens because embryos permeated with cryoprotectant can survive in faster freezing speeds.

The association between glycerol and sucrose was employed by Williams; Johnson¹⁴, which indicated the solution of 2.0M of glycerol plus 0.5M of sucrose as being the best combination, presenting 84% of embryo development.

The interruption of temperature decrease around –30ºC to –35ºC presents a good equilibrium between cell dehydration and intracellular ice formation, resulting in high embryonic survival rates during thawing⁸.

Different sucrose concentrations were added to the cryoprotectant solution with 10% of glycerol to cause embryo dehydration before freezing. This is necessary because at 0.3ºC/minute or 0.5ºC/minute some intracellular water remains, what causes intracellular ice formation and consequently embryo damage when plunged in liquid nitrogen at –24ºC or –32ºC.

The addition of 0.1% honeybee royal jelly to the cryoprotectant solution had the objective of enhancing the embryo survival rates. Bürkle et al.³ mentioned that these substances reduce the non lethal damage to the embryos during the freezing process. This may be due to the stabilization of the cell membrane noticed in the implantation and fetuses rates at –24ºC (Tab. 2 and 3).

The successfully frozen embryos may suffer irretrievable damage during cryoprotectant removal⁷, in this study, the cryoprotectant solution was removed stepwise^3,9, at a 22ºC temperature, using a 10% sucrose solution added to decreased glycerol concentration (6.6%; 3.3% and 0.0%). Excellent results were noted on embryo recovery and development rate after culture (Tab. 2 and 3).

The addition of sucrose in the solution containing decreased glycerol concentration or not^6,10 allows good diffusion of the cryoprotectant and controls the increase of cellular volume during cryoprotectant removal. The ideal concentration of sucrose for cryoprotectant removal is dependent on cryoprotectant concentration during freezing¹².

The efficiency of the freezing, thawing and cryoprotectant removal protocols in mice embryos may be evaluated by in vitro (Tab. 1) and in vivo development (Tab. 2 to 5).

Groups that have less sucrose in the cryoprotectant solution during freezing until –24ºC showed the worst results of embryo development (Tab. 1). This is probably because of excessive intracellular ice formation, due to insufficient dehydration leading membrane damage and the destruction of the cytoskeleton of embryo cells⁴.

The addition of 10% sucrose to the solution of 10% glycerol presented the greatest in vitro embryo development rate at 0.3ºC/minute and plunging temperature –24ºC (Tab. 1).

The in vivo development of embryos frozen at 0.3ºC/minute until –24ºC in the solutions with 10% glycerol and 10% glycerol + 0.1% honeybee royal jelly (Tab. 2) was similar. However, until –32ºC the implantation and fetuses rates were better in the absence of honeybee royal jelly (Tab. 4), in opposition to Bürkle et al.³ and Visintin et al.¹³.

Embryos frozen at 0.3ºC/minute and 0.5ºC/minute until –24ºC in solutions with 10% glycerol + 10% sucrose and 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly (Tab. 2 and 3) presented equal implantation rates and larger fetuses rates with honeybee royal jelly. This result was achieved notwithstanding¹³ not finding difference at the 0.3ºC/minute.

For the embryos frozen until –32ºC the influence of honeybee royal jelly was noted on the implantation rates at both speeds (Tab. 4 and 5). However, for the fetuses rates influence was noted at 0.5ºC/minute (Tab. 5) and not at 0.3ºC/minute (Tab. 4) agreeing with Visintin et al.¹³.

The honeybee royal jelly in the solution of 10% glycerol did not present influence on implantation and fetuses rates of embryos frozen at 0.3ºC/minute and plunging temperature of –24ºC and –32ºC.

CONCLUSIONS

The honeybee royal jelly in the solution of 10% glycerol and 10% sucrose influenced positively the implantation and fetuses rates of embryos frozen at 0.3ºC/minute and 0.5ºC/minute and plunging temperature of –24ºC.

The association of glycerol, sucrose and honeybee royal jelly resulted in excellent in vivo development rates, being recommended for freezing embryos at 0.3ºC/minute and 0.5ºC/minute, plunging temperature of –24ºC and quick thawing.

RESUMO

Embriões de camundongos foram congelados em 10% de glicerol adicionado de várias concentrações de sacarose e/ou 0,1% de geléia real, nas velocidades de 0,3ºC ou 0,5ºC/minuto até –24ºC ou –32ºC e descongelados em água a 22ºC durante 20 segundos. A diluição dos crioprotetores foi realizada em três etapas e o cultivo dos embriões no meio de Whitten em estufa com 5% de CO₂, 100% de umidade e 37ºC por 72 horas. O desenvolvimento in vitro até blastocisto variou entre 56,6% e 93,4%, mostrando que 10% de sacarose + 10% de glicerol foi eficiente na congelação. Blastocistos expandidos oriundos de embriões congelados até -24 ou –32ºC à velocidade de 0,3ºC/minuto em soluções contendo 10% de glicerol + 10% de sacarose; 10% de glicerol + 10% de sacarose + 0,1% de geléia real; 10% de glicerol + 0,1% de geléia real ou 10% de glicerol, transferidos para receptoras pseudoprenhes, apresentaram, respectivamente, taxas de fetos viáveis de 28,1% e 13,6%, 48,7% e 31,9%, 28,6% e 13,2%, 20,0% e 42,4%. Embriões congelados até –24ºC ou –32ºC a 0,5ºC/minuto nas soluções de 10% de glicerol + 10% de sacarose ou 10% de glicerol + 10% de sacarose + 0,1% de geléia real apresentaram, respectivamente, taxas de fetos viáveis de 29,0% e 15,3%, 48,8% e 32,0%. Adição de sacarose e de geléia real ao meio de congelação contendo 10% de glicerol proporcionou maiores taxas de fetos viáveis a 0,3ºC e 0,5ºC/minuto e imersão em nitrogênio líquido a –24ºC.

UNITERMOS: Camundongos; Embriões; Criopreservação; Sacarose; Geléia real.

Received: 08/06/1999

Accepted: 26/04/2000

¹ Departamento de Reprodução Animal da Faculdade de Medicina Veterinária e Zootecnia da USP – SP

• ¹
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^* This work was supported by grant from CNPq nº 52/1773-96.2

Publication Dates

• Publication in this collection
  11 May 2001
• Date of issue
  2000

History

• Received
  08 June 1999
• Accepted
  26 Apr 2000