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Cryopreservation of preantral ovarian follicles in situ from domestic cats (Felis catus) using different cryoprotective agents
Theriogenology 66 (2006) 1664–1666 www.journals.elsevierhealth.com/periodicals/the
Cryopreservation of preantral ovarian follicles in situ from domestic cats (Felis catus) using different cryoprotective agents Ana Kelen F. Lima *, Alexandre R. Silva, Regiane R. Santos, Daniele M. Sales, Andreia F. Evangelista, Jose´. R. Figueiredo, Lu´cia D.M. Silva Laboratory of Carnivore Reproduction – PPGCV/UECE, Paranjana Ave. 1700, Itaperi, Fortaleza-Ceara´ 60740-000, Brazil
Abstract The objective of this investigation was to verify the structural characteristics of preantral follicles (PAF) of cat ovarian tissue after cryopreservation in 1.5 M glycerol or ethylene glycol, using a slow-freezing procedure. Ovaries (n = 10) from domestic cats were divided into fragments. One fragment was immediately preserved for classical histology (fresh control), and additional fragments were immersed in minimum essential medium plus 10% bovine fetal serum (MEM + BFS), or MEM + BFS supplemented with 1.5 M glycerol or ethylene glycol. The samples were frozen and plunged into liquid nitrogen. After 1 wk, the samples were thawed. A total of 600 PAF were evaluated. In the fresh control, there were 71.3% normal PAF. After thawing, the rates of normal PAF were 26.0, 39.3 and 58.0% for samples without cryoprotectant or with glycerol or ethylene glycol, respectively. We concluded that ethylene glycol was useful for the cryopreservation of feline PAF in situ. # 2006 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Cryoprotectant; Preantral follicles; Ovary; Cat
1. Introduction Ovarian preantral follicles (PAF) represent 90% of the follicular population. Methods for manipulation of oocytes enclosed in PAF have been developed for several species, with the objective to rescue the PAF for in vitro culture by preventing follicular atresia [1]. In addition, cryobiological techniques have been recently used to store PAF derived from cattle [2] and sheep [3]. Therefore, recovery, cryopreservation and culture of oocytes included in PAF would help in the conservation of several animal species, with the objective of maintaining biodiversity. Among 37 felid species, only the domestic cat (Felis catus) is not at risk of extinction. Due to the phylogenetic
* Corresponding author. E-mail address: [email protected] (A.K.F. Lima). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.02.014
similarities between domestic and non-domestic carnivore species, the former are used as an experimental model for the others [4]. However, there is a lack of information concerning the manipulation of feline oocytes enclosed in PAF. Most studies have focussed on PAF recovery [5,6], with little information regarding the cryopreservation of feline PAF, except in isolated form [7]. The objective of this investigation was to verify the structural characteristics of feline PAF after cryopreservation of ovarian tissue in 1.5 M glycerol or ethylene glycol, using a slow-freezing procedure. 2. Materials and methods 2.1. Ovary collection and initial processing Five adult queens were subjected to ovariohysterectomy at local veterinary clinics. Immediately after collection, each ovary pair (n = 5) derived from the
A.K.F. Lima et al. / Theriogenology 66 (2006) 1664–1666
same queen was sliced into 11 similar fragments (approximately 3 mm thick). One fragment was randomly selected as the fresh control; it was fixed in Carnoy and prepared for routine histological evaluation. The remaining 10 fragments were allocated to various experiments; only three were used into the present study. The three fragments were processed for cryopreservation, including one without cryoprotectant (control) and the two others with glycerol or ethylene glycol. Each treatment was repeated five times. 2.2. Freezing/thawing processes One fragment was processed for cryopreservation by immersing it in minimum essential medium (MEM) plus 10% bovine fetal serum (BFS); the two other fragments were immersed in MEM + BFS supplemented with either 1.5 M glycerol or ethylene glycol. The fragments were initially placed in cryotubes and equilibrated for 20 min at 20 8C. Thereafter, samples were transferred to a cryomachine, cooled at 2 8C/min to 7 8C, manually seeded, cooled at 0.3 8C/min to 33 8C, and plunged into liquid nitrogen. After 1 wk, fragments were thawed by removing cryotubes from the liquid nitrogen, exposing them to room temperature (28 8C) for 1 min, and then placing them in a water bath (37 8C for 4 min). For removal of the cryoprotectant, the samples were subjected to three washes with 5 mL MEM + BFS at 5-min intervals. Finally, the fragments were subjected to classical histological evaluation to determine the effect of the cryoprotective agents on PAF viability. 2.3. Histological analysis and follicle evaluation After fixation in Carnoy for 12 h, the fragments were embedded in paraffin and 7-mm sections were stained with Schiff’s periodic acid and hematoxylin. The sections were examined by light microscopy (400 magnification) and PAF were classified according to developmental stage (primordial, primary and secondary) [8]. Furthermore, on the basis of their morphology, follicles were classified as normal (containing intact oocyte and wellorganized granulosa cells without a pycnotic nucleus) or degenerated (containing a somewhat shrunken oocyte with a pycnotic nucleus and strongly eosinophilic cytoplasm, with normal or disorganized granulosa cells and low cellular density) [1]. 2.4. Statistical analysis Results were expressed as mean (S.D.). The effects of cryoprotectants on the percentage of normal and
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degenerated PAF were analyzed by a Student’s t-test; values were considered statistically significant when P < 0.05. 3. Results A total of 600 PAF were evaluated. In the fresh control, 71.3% (21.4 4.2) of normal PAF were observed. After thawing, the rate of normal PAF in ethylene glycol was 58.0% (17.4 6.1). However, relative to cryopreservation in ethylene glycol, there was a significant reduction in the rate of normal PAF in the absence of cryoprotectant and in those supplemented with glycerol, 26.0% (7.8 1.3) and 39.3% (11.8 5.7), respectively (no significant difference between these two groups). 4. Discussion The present study evaluated the effects of the cryopreservation of feline PAF in 1.5 M glycerol or ethylene glycol on the percentage of normal PAF. Based on morphological analyses, immature feline oocytes (enclosed in preantral follicles within ovarian tissue) can be successfully cryopreserved when 1.5 M ethylene glycol was used as a cryoprotectant. In a first attempt to freeze isolated feline PAF, some authors [9] used a slow-freezing procedure with dimethyl-sulfoxide (DMSO), and obtained only 12% intact PAF after thawing. Later, the same research team [7] compared DMSO and propanediol, for feline PAF freezing and reported a decrease in the number of follicles with intact oocyte and granulosa cells when compared with non-frozen follicles (control). Recently, slices from the cat ovarian cortex were frozen using 1.5 M ethylene glycol and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice. The percentage of surviving follicles after cryopreservation and transplantation (10%) was low when compared to fresh tissue [10]. In the present study, we endeavored to develop an efficient method for cryopreservation of feline PAF in situ; we concluded that ethylene glycol was an effective cryoprotective agent for this purpose. Acknowledgements The authors thank Dr. Ronaldo Morato (ProCarnivore Association) for intellectual support of this research. A.K.F. Lima was supported by grants from FUNCAP.
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A.K.F. Lima et al. / Theriogenology 66 (2006) 1664–1666
References [1] Silva JRV, Lucci CM, Carvalho FCA, Ba´o SN, Costa SHF, Santos RR, Figueiredo JR. Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro. Theriogenology 2000;54:809–22. [2] Schellander K, Peli J, Schmoll F, Brem G. Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes. Theriogenology 1994;42:909–15. [3] Amorim CA, Rodrigues APR, Rondina D, Gonc¸alves PBD, Figueiredo JR, Giorgetti A. Cryopreservation of ovine primordial follicles using dimethylsulfoxide. Fertil Steril 2003;79:682–6. [4] Pope CE, Keller GL, Dresser BL. In vitro fertilization in domestic and non-domestic cats including sequences of early nuclear events, developments in vitro, cryopreservation and successful intra- and interspecies embryo transfer. J Reprod Fertil 1993;47:189–201. [5] Jewgenow K, Stolte M. Isolation of preantral follicles from nondomestic cats—viability and ultrastructural investigations. Anim Reprod Sci 1996;44:183–93.
[6] Lima AKF, Bezerra MB, Oliveira LC, Figueiredo JR, Silva LDM. Isolamento e caracterizac¸a˜o de folı´culos ovarianos pre´antrais em gatas dome´sticas (Felis catus). Rev Braz Reprod Anim 2003;27:396–7. [7] Jewgenow S, Penfold LM, Meyer HH, Wildt DE. Viability of small preantral ovarian follicles from domestic cats after cryoprotectant exposure and cryopreservation. J Reprod Fertil 1998;112:39–47. [8] Hulshof SCJ, Figueiredo Jr, Beckers JF, Bevers MM, Van Den Hurk R. Isolation and characterization of preantral follicles from foetal bovine ovaries. The Vet Quarterly 1994;16: 78–80. [9] Jewgenow K, Go¨ritz F. The recovery of preantral follicles from ovaries of domestic cats and their characterization before and after culture. Anim Reprod Sci 1995;39: 285–97. [10] Bosch P, Hernandez-Fonseca HJ, Miller DM, Wininger JD, Massey JB, Lamb SV, Brackett BG. Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice. Theriogenology 2004;61: 581–94.
Cryopreservation of preantral ovarian follicles in situ from domestic cats (Felis catus) using different cryoprotective agents Ana Kelen F. Lima *, Alexandre R. Silva, Regiane R. Santos, Daniele M. Sales, Andreia F. Evangelista, Jose´. R. Figueiredo, Lu´cia D.M. Silva Laboratory of Carnivore Reproduction – PPGCV/UECE, Paranjana Ave. 1700, Itaperi, Fortaleza-Ceara´ 60740-000, Brazil
Abstract The objective of this investigation was to verify the structural characteristics of preantral follicles (PAF) of cat ovarian tissue after cryopreservation in 1.5 M glycerol or ethylene glycol, using a slow-freezing procedure. Ovaries (n = 10) from domestic cats were divided into fragments. One fragment was immediately preserved for classical histology (fresh control), and additional fragments were immersed in minimum essential medium plus 10% bovine fetal serum (MEM + BFS), or MEM + BFS supplemented with 1.5 M glycerol or ethylene glycol. The samples were frozen and plunged into liquid nitrogen. After 1 wk, the samples were thawed. A total of 600 PAF were evaluated. In the fresh control, there were 71.3% normal PAF. After thawing, the rates of normal PAF were 26.0, 39.3 and 58.0% for samples without cryoprotectant or with glycerol or ethylene glycol, respectively. We concluded that ethylene glycol was useful for the cryopreservation of feline PAF in situ. # 2006 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Cryoprotectant; Preantral follicles; Ovary; Cat
1. Introduction Ovarian preantral follicles (PAF) represent 90% of the follicular population. Methods for manipulation of oocytes enclosed in PAF have been developed for several species, with the objective to rescue the PAF for in vitro culture by preventing follicular atresia [1]. In addition, cryobiological techniques have been recently used to store PAF derived from cattle [2] and sheep [3]. Therefore, recovery, cryopreservation and culture of oocytes included in PAF would help in the conservation of several animal species, with the objective of maintaining biodiversity. Among 37 felid species, only the domestic cat (Felis catus) is not at risk of extinction. Due to the phylogenetic
* Corresponding author. E-mail address: [email protected] (A.K.F. Lima). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.02.014
similarities between domestic and non-domestic carnivore species, the former are used as an experimental model for the others [4]. However, there is a lack of information concerning the manipulation of feline oocytes enclosed in PAF. Most studies have focussed on PAF recovery [5,6], with little information regarding the cryopreservation of feline PAF, except in isolated form [7]. The objective of this investigation was to verify the structural characteristics of feline PAF after cryopreservation of ovarian tissue in 1.5 M glycerol or ethylene glycol, using a slow-freezing procedure. 2. Materials and methods 2.1. Ovary collection and initial processing Five adult queens were subjected to ovariohysterectomy at local veterinary clinics. Immediately after collection, each ovary pair (n = 5) derived from the
A.K.F. Lima et al. / Theriogenology 66 (2006) 1664–1666
same queen was sliced into 11 similar fragments (approximately 3 mm thick). One fragment was randomly selected as the fresh control; it was fixed in Carnoy and prepared for routine histological evaluation. The remaining 10 fragments were allocated to various experiments; only three were used into the present study. The three fragments were processed for cryopreservation, including one without cryoprotectant (control) and the two others with glycerol or ethylene glycol. Each treatment was repeated five times. 2.2. Freezing/thawing processes One fragment was processed for cryopreservation by immersing it in minimum essential medium (MEM) plus 10% bovine fetal serum (BFS); the two other fragments were immersed in MEM + BFS supplemented with either 1.5 M glycerol or ethylene glycol. The fragments were initially placed in cryotubes and equilibrated for 20 min at 20 8C. Thereafter, samples were transferred to a cryomachine, cooled at 2 8C/min to 7 8C, manually seeded, cooled at 0.3 8C/min to 33 8C, and plunged into liquid nitrogen. After 1 wk, fragments were thawed by removing cryotubes from the liquid nitrogen, exposing them to room temperature (28 8C) for 1 min, and then placing them in a water bath (37 8C for 4 min). For removal of the cryoprotectant, the samples were subjected to three washes with 5 mL MEM + BFS at 5-min intervals. Finally, the fragments were subjected to classical histological evaluation to determine the effect of the cryoprotective agents on PAF viability. 2.3. Histological analysis and follicle evaluation After fixation in Carnoy for 12 h, the fragments were embedded in paraffin and 7-mm sections were stained with Schiff’s periodic acid and hematoxylin. The sections were examined by light microscopy (400 magnification) and PAF were classified according to developmental stage (primordial, primary and secondary) [8]. Furthermore, on the basis of their morphology, follicles were classified as normal (containing intact oocyte and wellorganized granulosa cells without a pycnotic nucleus) or degenerated (containing a somewhat shrunken oocyte with a pycnotic nucleus and strongly eosinophilic cytoplasm, with normal or disorganized granulosa cells and low cellular density) [1]. 2.4. Statistical analysis Results were expressed as mean (S.D.). The effects of cryoprotectants on the percentage of normal and
1665
degenerated PAF were analyzed by a Student’s t-test; values were considered statistically significant when P < 0.05. 3. Results A total of 600 PAF were evaluated. In the fresh control, 71.3% (21.4 4.2) of normal PAF were observed. After thawing, the rate of normal PAF in ethylene glycol was 58.0% (17.4 6.1). However, relative to cryopreservation in ethylene glycol, there was a significant reduction in the rate of normal PAF in the absence of cryoprotectant and in those supplemented with glycerol, 26.0% (7.8 1.3) and 39.3% (11.8 5.7), respectively (no significant difference between these two groups). 4. Discussion The present study evaluated the effects of the cryopreservation of feline PAF in 1.5 M glycerol or ethylene glycol on the percentage of normal PAF. Based on morphological analyses, immature feline oocytes (enclosed in preantral follicles within ovarian tissue) can be successfully cryopreserved when 1.5 M ethylene glycol was used as a cryoprotectant. In a first attempt to freeze isolated feline PAF, some authors [9] used a slow-freezing procedure with dimethyl-sulfoxide (DMSO), and obtained only 12% intact PAF after thawing. Later, the same research team [7] compared DMSO and propanediol, for feline PAF freezing and reported a decrease in the number of follicles with intact oocyte and granulosa cells when compared with non-frozen follicles (control). Recently, slices from the cat ovarian cortex were frozen using 1.5 M ethylene glycol and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice. The percentage of surviving follicles after cryopreservation and transplantation (10%) was low when compared to fresh tissue [10]. In the present study, we endeavored to develop an efficient method for cryopreservation of feline PAF in situ; we concluded that ethylene glycol was an effective cryoprotective agent for this purpose. Acknowledgements The authors thank Dr. Ronaldo Morato (ProCarnivore Association) for intellectual support of this research. A.K.F. Lima was supported by grants from FUNCAP.
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A.K.F. Lima et al. / Theriogenology 66 (2006) 1664–1666
References [1] Silva JRV, Lucci CM, Carvalho FCA, Ba´o SN, Costa SHF, Santos RR, Figueiredo JR. Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro. Theriogenology 2000;54:809–22. [2] Schellander K, Peli J, Schmoll F, Brem G. Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes. Theriogenology 1994;42:909–15. [3] Amorim CA, Rodrigues APR, Rondina D, Gonc¸alves PBD, Figueiredo JR, Giorgetti A. Cryopreservation of ovine primordial follicles using dimethylsulfoxide. Fertil Steril 2003;79:682–6. [4] Pope CE, Keller GL, Dresser BL. In vitro fertilization in domestic and non-domestic cats including sequences of early nuclear events, developments in vitro, cryopreservation and successful intra- and interspecies embryo transfer. J Reprod Fertil 1993;47:189–201. [5] Jewgenow K, Stolte M. Isolation of preantral follicles from nondomestic cats—viability and ultrastructural investigations. Anim Reprod Sci 1996;44:183–93.
[6] Lima AKF, Bezerra MB, Oliveira LC, Figueiredo JR, Silva LDM. Isolamento e caracterizac¸a˜o de folı´culos ovarianos pre´antrais em gatas dome´sticas (Felis catus). Rev Braz Reprod Anim 2003;27:396–7. [7] Jewgenow S, Penfold LM, Meyer HH, Wildt DE. Viability of small preantral ovarian follicles from domestic cats after cryoprotectant exposure and cryopreservation. J Reprod Fertil 1998;112:39–47. [8] Hulshof SCJ, Figueiredo Jr, Beckers JF, Bevers MM, Van Den Hurk R. Isolation and characterization of preantral follicles from foetal bovine ovaries. The Vet Quarterly 1994;16: 78–80. [9] Jewgenow K, Go¨ritz F. The recovery of preantral follicles from ovaries of domestic cats and their characterization before and after culture. Anim Reprod Sci 1995;39: 285–97. [10] Bosch P, Hernandez-Fonseca HJ, Miller DM, Wininger JD, Massey JB, Lamb SV, Brackett BG. Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice. Theriogenology 2004;61: 581–94.