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Cryopreservation of bos taurus vs bos indicus embryos: are they really different?
ELSEVIER
CRYOPRESERVATION OF BOS TAURUS VS BOS INDICUS EMBRYOS: ARE THEY REALLY DIFFERENT? J.A. Visintin’, J.F.P. Martins’, E.M. Bevilacqua*, M.R.B. Mello’, A.C. Nicacio’, M.E.O.A. AssumpcZo’ ‘Department of Animal Reproduction, Faculty of Veterinary Medicine, *Department of Histology and Embryology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, 05508-000, Brazil ABSTRACT Cryopreservation with storage at very low temperatures is essential to make full use of this technology for both biological and commercial reasons. However, most mammalian cells will die if exposed to these temperatures unless they are exposed to cryoprotectant solutions and cooled and warmed at specific rates. Lowering temperature below 0°C introduces the risk of intracellular ice formation, which likely increases rapidly as the temperature falls. Evidence indicates that ice formation during cooling can cause significantly more damage than ice formation during warming. Comparisons of the toxicity of various cryoprotectants indicated that ethylene glycol (EG) is a nontoxic compound for murine and bovine embryos. The 3.6 M EG solution resulted in similar high survival rates when compared with nonfrozen embryos; deleterious effects of high concentrations of EG became apparent at 7.2 M. The use of EG provides a nontoxic method for the rapid and simplified controlled freezing of in vivo bovine compact morulae-early blastocyst, avoiding the risk of injury caused by high concentrations of cryoprotectants usually required for vitrification. However, in vivo embryos used for freezing and thawing require further studies to understand the ultrastructural changes during the freezing procedure with EG as the single cryoprotectant, especially between Holstein and Nelore cows. This paper describes the ultrastructure of bovine compact morulae-early blastocysts derived by in vivo methods from Holstein and Nelore cows to investigate the fresh morphology as well as that after exposure to cryoprotectant and cryopreservation by conventional slow freezing, quick freezing (nitrogen vapor), and vitrification. Q 2001 by Elsevier Science Inc. INTRODUCTION When cells are frozen, they are subjected to stress resulting from water-solute interactions that arise through ice crystallization. Crystallization induces unfrozen pocket formation of hyperosmotic solution while cooling progresses to approximately -50°C (6). This process results in withdrawal of intracellular water, subsequent cell shrinkage, and possible influx of ions (1, 7). Thawing involves a reversal of these effects, and the consequent inward water flux may cause cell membrane disruption. Because overly rapid freezing causes lethal intracellular ice formation, the optimal cooling rate is thought to be slow enough to prevent this lethal effect, but fast enough to minimize the harmful effects of prolonged exposure to high salt concentrations (“solution effects”). Acknowledgements: The author wishes to acknowledge Dr. Matthew B. Wheeler and Dr. Eric M. Walters for the review in the manuscript. Research was supported by FAPESP. Theriogenology 57345-359. 0 2001 Elsevier Science Inc.
2002
0093-691X/02/$-see front matter PII: SOO93-691X(01)00675-6
Theriogenology
Cryopreservation involves exposure to non-physiologically low temperatures even before freezing occurs. This process is known to induce changes in two-dimensional membrane lipid organization or “packing” (lipid phase transitions) and, in turn, to modify the kinetic properties of intramembranous enzymes (3, 4, 5). Efforts to correlate susceptibility to cryoinjury with the membrane lipid composition among species have suggested that cold shock, a phenomenon by which cryoinjury is induced by sudden cooling without freezing, is more severe when cell membrane sterol concentrations are low and polyunsaturated fatty acid concentrations are high (238). Cryopreserved embryos are widely used in assisted reproduction of domestic animals. Their value in assisted reproduction in cattle is clearly illustrated by the large number of embryos that are frozen and transferred each year. Cryopreservation with storage at very low temperature (e.g., liquid nitrogen at -196’C) is essential to make full use of this technology for both biological and commercial reasons, as it can simultaneously reduce the costs, genetic drift, and disease that are normally associated with maintaining live animals and cell lines. However, most mammalian cells will die if exposed to these temperatures unless they are exposed to cryoprotectant solutions and cooled and warmed at specific rates. Despite considerable difficulties, several effective cryopreservation protocols have been developed for oocytes and embryos of the mouse (13, 22, 26,27) and cow (18,28), and the knowledge gained from this research is starting to be applied to other species. Damage is most likely to occur at temperatures between +15 and -9O’C. Lowering temperature below O’C introduces the risk of intracellular ice formation, which likely increases rapidly as the temperature falls (39). To prevent intracellular ice formation or minimize the damage, all commonly used cryopreservation protocols are designed to dehydrate cells. They usually also achieve very high extra cellular solute concentrations. With regard to slow cooling, this is achieved by placing the cells in a solution containing 10 to 11% (v/v) penetrating cryoprotectant (approximately 1.5 M). The temperature is then lowered, and ice crystal growth is initiated (seeded) within the solution. As the ice crystals grow, the water in the solution is converted from its liquid to a solid state. This increases the concentration of the solutes, which draws water out of the cells. The lower the temperature, the more water can be incorporated into ice, but the rate at which water can leave a cell also falls as the ambient temperature is lowered. The success of slow cooling therefore depends on achieving the optimal balance (equilibrium) between the rate at which water can leave the cell and the rate at which it is converted into ice. Most procedures currently used to cryopreserve embryos, oocytes, and ovarian tissue stipulate a cooling rate of 0.3 to O.S”C/min from the seeding temperature (usually -5 to -9“C) down to a lower temperature, usually between -33 and -4O”C, after which they can be placed in liquid nitrogen. Although cells cooled in this manner still contain some water at the time they are placed in the liquid nitrogen, there is not enough intracellular water to cause damage, provided that appropriate rapid warming protocols are used. Most rapid cooling protocols use solutions with high concentrations of solutes (e.g., cryoprotectants and sugars), which rapidly draw water out of the cells (26,28). In these solutions, cells become sufficiently dehydrated and permeated by cryoprotectants to tolerate direct immersion into liquid nitrogen or nitrogen vapor. These are commonly referred to as nonequilibrium procedures. The non-equilibrium procedures are subdivided into two categories depending on whether or not ice forms in the solution during cryopreservation. A number of solutions are formulated so that they solidify without any ice crystal formation. These are glassforming or “vitrifying” solutions. When measurable or visible amounts of ice form during either
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cooling or warming, the procedures should be noted as rapid cooling (or ultra-rapid cooling) (26). Both the cooling and warming rates, as well as the composition and concentration of the solution will influence how much, if any, ice will form (15,26,28). Evidence indicates that ice formation during cooling can cause significantly more damage than ice formation during warming. Cooling can also disrupt cells in other ways. Oocytes of many, but not all species, are damaged or killed by short-term exposure to temperatures between +15”C and 0°C. This is best documented in pig and cow oocytes and embryos (9, 18, 19,28). The damage may be correlated with the large amount of dark lipid-like material found in these species because lipid removal or lipid polarization reduces chill and cryoinjury (23). Slow cooling procedures can be highly detrimental to oocytes of chill-sensitive species, as they involve prolonged exposure to the temperatures at which most damage occurs, e.g., with the use of a high equilibration temperature and rapid or very rapid cooling rates, good results for oocytes of chill sensitive species can be achieved (12, 17, 19, 28). Strategies to obtain very rapid cooling rates include the use of copper grids, open pulled straws, wire loops, and microdrops on straws sides and foil (16, 19,28). These very rapid cooling rates benefit embryos and oocytes by providing a very fast transit through the temperature zone, which causes damage to embryos and oocytes; it also facilitate vitrification at lower, and hence less toxic, cryoprotectant concentrations. The choice of buffer system, nature of cryoprotectants, and additives such as sugars, calcium chelators, and antioxidants have all been shown to influence cell survival profoundly. It is unlikely that the fundamental role of these substances is to modify the water permeability of the cell membrane; therefore, other explanations for their different and species-species effects must be sought. Cryoprotectant mixtures (e.g., EG and DMSO) may have some advantages over solutions containing only one penetrating cryoprotectant (11,24,28). However, the overall composition of the solution is also important, as embryo and oocyte survival is modified by other components, including sugars, macromolecules, or polymers (10,20,2 1, 22); EGTA (29); and salts (27). High concentrations of polymers such as Ficoll and Dextran (e.g., >30%) appear to be non-toxic to the embryos and can be used to replace an approximately equal amount of ethylene glycol (by weight) without changing the solution’s glass transition temperature (15, 25). It has already been shown (2 1) that low concentrations of Dextran (1 mg/mL) do not benefit oocytes rapidly cooled in 6 M DMSO, but solutions containing only 15% EG (together with 40% Ficoll) allow effective cryopreservation of mouse embryos (14, 15). It now needs to be ascertained whether these polymer-based solutions are suited to oocytes or embryos of other species. The cryoprotectant EG has proved to be nontoxic for murine embryos (4, 36) and may improve the survival rate of frozen and thawed bovine embryos (33, 34, 35, 45). The high penetration rate and the low toxicity of this cryoprotectant allow direct transfer after thawing without preceding dilution (31, 37). Ethylene glycol has been successfully employed at concentrations of 1.5-l .8 M for the freezing of in vivo bovine embryos (30,3 1,32,37). The protocol employing EG at a concentration of 3.6 M was used for successful cryopreservation of IVP bovine embryos in controlled freezing (35). The rapid freezing (nitrogen vapor) of in vivo mouse embryos (40) has been applied with similar success. The rapid penetration of the embryos by EG seems to avoid preferential cryoinjury of sensitive trophoblasts
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and thus allows high survival rates of IVP-expanded blastocysts. The different survival rates following freezing can be attributed to variation in the type and concentration of employed cryoprotectants. Comparisons of the toxicity of various cryoprotectants indicated that EG is a nontoxic compound for murine (36, 38, 40) and bovine (45) embryos. The 3.6 M EG solution resulted in similar high survival rates when compared with nonfrozen embryos; deleterious effects of high concentrations of EG became apparent at 7.2 M. (35). However, when the concentration of EG is gradually increased, embryos tolerate even a final EG concentration of 7.2 M (38,40,41,42). It cannot be ruled out that the direct dilution of the high EG concentration has been deleterious attributable not to the toxicity of the solution but to extreme osmotic stress, resulting in poor survival rates. It has been shown, however, that bovine IVP embryos tolerate direct dilution of a vitrification solution consisting of 40% EG, 6% polyethylene glycol, and 0.5 M sucrose without a decrease in viability (43). Posthaw direct dilution of 1.5 M EG from early mouse embryos was equally efficient as was the dilution with sucrose (40,44). This suggests that the use of sucrose for cryoprotectant dilution is not required to maintain embryonic developmental capacity. The use of EG provides a nontoxic method for the rapid and simplified controlled freezing of in vivo bovine compact morulae-early blastocyst, avoiding the risk of injury caused by high concentrations of cryoprotectants usually required for vitrification. However, in vivo embryos used for freezing/thawing need further studies to understand the ultrastructural changes during the freezing procedure with EG as the single cryoprotectant, especially between Holstein and Nelore cows. The aim of this study was to describe the ultrastructure of bovine compact morulae-early blastocysts derived by in vivo methods from Holstein and Nelore cows and to investigate the fresh morphology as well as that after exposure to cryoprotectant and cryopreservation by conventional slow freezing, quick freezing (nitrogen vapor), and vitrification. MATERIALS AND METHODS Excellent morulae and early blastocysts from Holstein and Nelore cows were produced by in vivo methods and distributed into three groups 1) fresh, 2) exposed to cryoprotectant, and 3) cryopreserved by conventional slow freezing, quick freezing, and vitritiction groups (Table 1). Table 1. Distribution of Nelore and Holstein embryos in fresh, exposed to cryoprotecnt, and freezing group categories. Embryos _.____ __ .-..IProtocols .._, ____.__-. . . . .-.. . -.. . -. .PBS + 0.4 % BSA Fresh 1.8 MEGa Exposed 3.0 M EG + 0.3 M sucrose 7.2 M EG + 0.3 M sucrose+ 18% Ficoll 1.8 M EG - controlled freezing at -0.5 Umin Frozen 1.8 M EG - controlled freezing at -1.2 C/min 3.0 M EG + 0.3 M sucrose - quick freezing 7.2 M EG + 0.3 M sucrose + 18% Ficoll -vitrification “EG =ethylene glycol.
Holstein ...-.- . Nelore ...- - . 3 3 2 2 3 3 1 3 2 3 3 3 3 3 1 2
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To investigate the toxicity of cryoprotectants, embryos were exposed to three different cryopreservation methods: 1) 1.8 M EG for 10 min; 2) 3 M EG + 0.3 M sucrose for 5 min, and 3) pre-equilibration in 3.6 M EG for 3 min and 7.2 M EG + 0.3 M sucrose + 18% Ficoll (MW 70.000) for 1 min. Subsequently, the embryos were washed three times in PBS + 0.4% BSA and prepared for evaluation by light and transmission electron microscopy (Table 1). To investigate cryoinjury, the embryos were exposed to the cryoprotectant and loaded in straws, which contained three columns (in both extremities with PBS + 0.4% BSA and in the middle embryo + cryoprotectant), and frozen. The columns were as follows. 1) 1.8 M EG. The straws were placed at -7°C into a metanol freezer (Haacke, Germany), and seeding was induced with super-cooled forceps after 5 min of equilibration. The samples were cooled at O.S”C/min or 1.2’C/min to -3 l°C and then plunged into liquid nitrogen. 2) 3 M EG. The straws were placed at nitrogen vapor (-17O’C) for 2 min and plunged into liquid nitrogen. 3) 7.2 M EG. The straws were plunged directly in liquid nitrogen. After storage for 1 wk, the straws were thawed at room temperature (22’C) for 10 set in air and in 25“C water bath for 20 sec. The cryoprotectant was diluted inside the straw for 5 min and, after that, the embryos were washed three times in PBS + 0.4% BSA. After thawing and rehydration, the embryos were prepared for evaluation by light and transmission electron microscopy. Embryos were fixed in 2.5% glutaraldehyde in 0.1 M PBS with 0.05 M sucrose for at least 1 hr, removed from fixative, and washed three times for a total of 15 min in 0.1 M PBS. Individual embryos were embedded in 2% agar and then removed in 3-mm3 blocks. Agarose blocks were postfixed in 2% osmium tetroxide (0~04) in 0.1 M PBS for 1 hr, washed three times in 0.1 M PBS for a total of 15 min, dehydrated through ascending concentrations of alcohol, and embedded in Spurr resin. Resin blocks were solidified at 60°C. Semi-thin sections (2 urn) had been stained with toluidine blue for light microscopy. Ultrathin sections (80 nm) from each blastocyst were collected on copper grids and post-stained with uranyl acetate and examined in a transmission electron microscopy (Table 1).
RESULTS AND DISCUSSION Morphology of Non-Frozen (Fresh) Embryos Fresh embryos (n =6) showed discoidal form delimited by the zona pellucida (ZP). In the compact morulae-early blastocyst, the pellucida zone thickness measured 15 pm and, in both cases, presented homogeneous texture in the internal face and more porous aspect in the external faces. The disposition of blastomeres are similar in Nelore and Holstein embryos. The layer of trophoblastic cells in the blastocysts and the layer of embryonic cells in contact with the ZP in morulae revealed elongated form and adhered cells, reflective of a typical epithelium [Figure l(A and C)]. In the blastocyst, the trophoblastic cells surrounded the blastocoele cavity and the inner cell mass. The cells of inner cell mass presented irregular star-like appearance, maintaining spaces between them but establishing occasional junctions [Figure l(B)].
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This arrangement and disposal of the blastomeres in this phase of the development also were reported by other authors, independent of the bovine breed considered (46 47, 48, 53, 54, 55). In the morulae, between the peripheral cells and those disposed more internally, cavities of varied dimensions could be found [Figure l(A and B)]. This finding probably relates to the process of liquid accumulation after the compactation phase and subsequent organization of blastocoele, which increases with embryonic development. Pedersen and Spindle (49), using mouse embryos, demonstrated that the accumulation of fluid in blastocoele is essential to blastomere differentiation. In fact, the cells more peripheral (those that surround these cavities) become more flattened, electrondense, and have an organele content different from the others, indicating that the peripheral blastomere differentiation in trophoblastic cells has been taken. Most of the blastomeres’ nuclei are central, presenting a predominance of eucromatine, one or two proeminent nucleolus. In the blastocysts, the trophoblastic cells revealed a match between themselves by GAP junctions, consisting mainly of tight junctions and desmosomes, which was also observed by other authors (56). Interdigital projections were also observed between the membranes of contiguous cells [Figures l(B and D)]. In the free surface, these cells showed a great amount of microvilus and vesicles covered in the apical region, which indicates an extensive contact surface with the perivitelline space (PvS) specialized in the molecule captation [Figure l(F)]. In this place, cellular debris is frequently found [Figure l(D and F)]. Cells with degenerative features and cellular death were observed similarly in the ICM and in the layer of trophoblastic cells [Figure l(B)]. Other authors had similar findings in bovine (54), mouse (50) equine (56), and monkey (51); those authors categorized these finding as important events for normal embryo development. Enders et al. (52) suggested that, during embryonic differentiation, there is a regulation in the number of blastomeres. This regulation is based on the capacity of the embryo to remove cells that exhibit reduction in its potential development. The ultra-structural morphology and distribution of blastomere cellular compounds showed similarity in the embryos among breeds, which corroborate with the descriptions carried through Crosier et al. (48) and Fair et al. (55). Great numbers of ribosomes and poliribosomes prevailed in the cytoplasm of these cells, as compared with other organeles. Differences, however, had been observed in the pattern and concentration of the lipid droplets, similar to endosomics, phagocytics vacuoles, and mitochondria structures (Table 2). The lipid droplets were larger and in greater number in the Holstein embryos. Vesicles of variable dimensions with contents of different electrondensities that presented partially digested material were found in the blastomeres, showing a predominance of this phenomena in Nelore [Figure l(E)]. These structures appear to form fagosomic vacuoles with or without the secondary fusing of lisosomes. Rounded and prolongated mitochondria with transversal crystals were more abundant in the cells of Nelore embryos. Differences in these cellular components between Bos taurus taurus and Bos taurus indicus embryos had also been found by Esper and Barbosa (57); however, little literature of regarding this is well known. Table 2. Morphologic differences among Nelore and Holstein embryos.a Holstein Characteristics ---. .^.-._..___ . Nelore -----._._..._--..--..-_._.-. ++ + Mitochondria ++ +++ Lipid droplets +++ ++ Endosomic cistern vesicles “The values had been estimated on the basis of morphologic analysis: + (few), ++ (intermediate values), and +++ (many).
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Morphology of Exposed Embryos to the Cryoprotectants The protocols of cryopreservation in both breeds revealed low toxicity to the embryos that, morphologically, presented similar features compared with fresh embryos. The few differences observed in relation to cryopreservation refer to a larger PvS where the distance between ZP and blastomeres increases [Figure 2(A)]. Eventually, deformities in relation to the original form of the blastocysts were observed [Table 3 and Figure 2(B)]. According to Dias et al. (61) and Alvarenga (56), who analyzed bovine embryos, the increase of PvS refers to the incomplete re-expansion of the blastomeres during the rehydratation of the cells, which is associated with cryoprotectant removal. Table 3. Alterations caused by exposure of Nelore and Holstein embryos to the cryoprotectants.a Alterations observed after cryoprotectant exposure ..- - - . --_.- . --__-.-.. .- ._._ -... ..___.._.._ . . .-....Increase of the perivitelline space Alterations in the embryo form “(+) present / (-) not present.
Nelore and Holstein Embryos ~. . . _..-._... ... -. .-_ .___. + +
Morphology of Frozen Embryos Although after thawing, the frozen embryos apparently kept their form and similar structure in comparison with the fresh embryos, a detailed analysis showed that the embryonic cells, mainly in Nelore, presented significant alterations. Independent of the protocol used for freezing and the origin of the embryo, embryonic cells presented qualitative and quantitative signs of degeneration and cellular death when compared with fresh embryos (Figure 3). These cells occupied the same areas as cells with normal appearance but showed mitose signs. One of the most common degenerative features was the swelling of cells with cytoplasmatic injuries and vacuolization nucleous [Figure 3(A and B)]. Cellular lysis was also observed, as well as cells with discontinuous plasmatic membranes [Figure 3(A)]. These findings are similar to those observed during the processes of cellular death at necrosis or in cells submitted to hyperosmotic stress, which are characteristic of alterations in permeability of membranes. Similar comments were also been reported by Alvarenga (56) when equine embryos were frozen. The author describes the process in terms of alterations in the distribution of lipids and proteins in the membrane, causing abnormal rearrangements of these molecules and subsequent changes in the cellular metabolism. Hinkorska-Galcheva (58) affirmed that modifications in the molecular organization of lipids in the membranes can cause structural and functional alterations of its enzymes and proteins. It is possible that our findings also reflect the deleterious action of the freezing and thawing process among blastomeres through alterations in permeability of plasmatic and organelle membranes. The presence of cellular debris was also observed in blastocoele; PvS; and, in some samples, in the intercellular space. The presence of great intra-embryonic cavities [Figure 3(A and C)] was also observed. Possibly, this cellular debris precedes the lysis process following the cryopreservation process. Morphologic features of the embryonic cells of Nelore and Holstein embryos were better preserved under control freezing than under quick freezing and the vitrification process. However, during the quick freezing and vitrification processes, Holstein embryos exhibited better morphologic conditions than did Nelore embryos. These results confirm
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the results of Zanenga (60) who showed that homogeneous behavior during the freezing process for Bos tam-us embryos, but not Bos indicus embryos, varied in quality after thawing, resulting in lower pregnancy rates. However, studies carried out by Rail, Fahy (59), and Rall et al. (62) demonstrated the viability of mouse embryos after devitrification (thawing). Rodrigues (63) related that vitrification results in bovine are unsatisfactory. Other significant findings were the sensible reduction of microvilus on the surface of the trophoblastic cells [Figure 3(E)] and an increase in the incidence of vesicles of the endosomic system, mitochondrial vacuolization [Figure 3(D)], and intense expansion of membrane compartment (endoplasmatic reticulum and/or Golgi apparatus).
Table 4. Alterations caused by different freezing processes on Nelore and Holstein embryos.a
Freezing protocols
-0.5’C
Nelore -1.2’C Vapor7%%s
-0.5’C
Holstein -1.2’C Vapor
Vitrif
Dead cells
++
++
+++
+++
+
+
++
++
Lysed cells
++
++
+++
++
+
+
++
++
Swollen cells
+++
+++
+++
+++
++
++
+++
+
Cellular debris
+
+
+++
+++
++
++
++
++
++
+
+
+++
+++
+++
++
+++
+++
++
++
++
++ ++ ++ ++ + Microvilus on trophoblastic surface +++ ++t +++ +++ +++ Vesicle of endossomic system ++ +++ +++ ++ ++ Mitochondrial vacuolization +++ + + + +++ Cistern enlargement of REG and Golgi aThe values had been estimated on the basis of morphologic analysis: + values), and +++ (many). bVitrif = vitrification.
(few), ++ (intermediate
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Figure t. Fresh embryos. A) Holstein embryo (zp = zona pellueida, tf = differentiation of trophoblastic cell, icm = inner cell mass, and * = cavities between icm and tf); LM × 1000. B ) N e l o r e embryo. Contiguous inner cells mass (de = degenerated cell arrows, interdigital projections); TEM x 3000. C) Nelore embryo; L M × 1000. D) Holstein embryo. Cytoplasmic detail ofthrophoblastic cell (pvs = perivitelline space, arrow = interdigital projection, black arrow = cellular debris in pvs, and * = large lipid droplets); TEM × 4000. E) Nelore embryo. Cytoplasmic detail ofthrophoblastic cell (N = nucleous; * = vesicles presenting a partially digested material); TEM × 10000. F) Holstein embryo. (arrow = microvilli in trophoblastic call surface, black arrow = cellular debris in pvs, and * = some lipid droplets); TEM x 2000.
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Figure 2. Embryos after cryoprotectant exposure. A) Nelore embryo showing cellular aspects; TEM x 1000. a) Insert of Nelore embryo after cryoprotectant exposure showing larger perivitelline space; LM × 400. B) Holstein embryo showing cellular aspects; TEM × 2000. b) Insert of Holstein embryo deformed after cryoprotectant exposure; LM × 400.
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Figure. 3. Frozen and thawed embryos. A) Nelore embryo with various cytoplasmatic injuries (zp = zona pellucida, pvs = perivitelline space, arrow = cellular debris in PvS, black arrow = cellular lysis, * = generated vacuolization, and + -- nuclear vacuolization); T E M x 1500. a) Insert o f Nelore embryo showing a similar structure to the fresh one; LM x 400. B) Holstein embryo with various cytoplasmatic injuries (N = nucleous, arrow - nuclear injuries, and * = cytoplasmatic vacuolization); T E M x 2000. b) Insert o f Holstein embryo showing a similar structure to the fresh one; LM x 400. C) Holstein embryo (N = some nucleous; * = great intra-embryonic cavities); T E M x 1200. D) Holstein embryo (arrow = mitochondrial vacuolization; * = intense expansion o f membranous compartment); T E M × 7500. E) Nelore embryo (N = nucleous; arrow = absence o f microvilli on surface of trophoblastic cell); T E M x 1500.
Theriogenology
In conclusion, Holstein embryos exhibit more intracellular lipid when compared with Nelore embryos. Embryos exposed to cryoprotectants demonstrated none or few morphological alterations in both breeds, indicating that embryonic cells were mildly affected by cryoprotectant toxicity. Holstein and Nelore embryos frozen by the slow freezing method had better morphological quality than quick freezing and vitrification. In the quick freezing and vitrification REFERENCES 1.
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Lane M, Forest KT, Lyons EA, Bavister BD. Live births following vitrification of hamster embryos using a novel containerless technique. Theriogenology 1999;5 1: 167 abst. Le Gall F, Massip A. Cryopreservation of cattle oocytes: effects of meiotic stage, cycloheximide treatment, and vitrification procedure. Cryobiology 1999;38:290-300. Martin0 A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultrarapid cooling. Biol Reprod 1996;54: 1059- 1069. Martin0 A, Pollard JW, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev 1996;45:503-5 12. Miyake T, Kasai M, Zhu SE, Sakurai T, Machida T. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol based solution by a simple method. Theriogenelogy 1993;40:121-134. O’Neil L, Paynter SJ, Fuller BJ, Shaw RW. Vitrification of mature mouse oocytes in dimethylsulphoxide-improved results following the addition of polyethylene glycol but not dextran. Cryo Lea 1998;19:141-146. O’Neil L, Paynter SJ, Fuller BJ, Shaw RW. Vitrification of mature mouse oocytes: improved results following addition of polyethylene glycol to a dimethyl sulfoxide solution. Cryobiology 1997;34:295-301. Otoi T, Yamamoto K, Koyama N, Tachikawa S, Murakami M, Kikkawa Y, Suzuki T. Cryopreservation of mature bovine oocytes followig centrifugation treatment. Cryobiology 1997; 34:36-41. Paynter SJ, Cooper A, Fuller BJ, Shaw RW. Cryopreservation of bovine ovarian tissue: structural normality of follicles after thawing and culture in vitro. Cryobiology 1999;38:301-309. Shaw J, Kuleshova L, McFarlane D, Trounson AO. Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll or sucrose. Cryobiology 1997;35:219-229. Shaw JM, Oranratnachai A, Trounson AO. Cryopreservation of oocytes and embryos. In: Trounson AO, Gardner D(eds), Handbook of In Vitro Fertilization. 2nd Edition, CRC Press, 1999;1-400. Stachecki JJ, Cohen J, Willadsen SM. Cryopreservation of unfertilized oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 1998;37:346-345. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: A new way to avoid cryoinjuries of mammalian ova and embryos Mol Reprod Devel 1998;51:53-58. Younis Al, Toner M, Albertini DF, Biggers JD. Cryobiology of non-human primate oocytes. Hum Reprod 1996; 11: 156-l 65. Bracke C, Niemann H. New aspects in the freezing of embryos from livestock. In Proceedings, 11th Reunion AETE. Hannover, 1995; 10 1- 111. Dochi 0, Imai K, Takahura H. Birth of calves after direct transfer of thawed bovine embryos stored frozen in ethylene glycol. Anim Reprod Sci 1995;38:179-185. McIntoch A, Hazeleger N L. The use of ethylene glycol for freezing bovine embryos. Theriogenology 1994;41:253 abst. Saha S, Suzuki T. Vitrification of in vitro produced bovine embryos at different ages using one-and three-step addition of cryoprotective addition. Reprod Fertil Dev 1997;9:74 l-746. Saha S, Otoi T, Takagi M, Boediono A, Sumantri C, Suzuki T. Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, trehalose and polyvinylpyrrolidone. Cryobiology 1996;33:291-299.
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Esper CR. Avaliacgo dos efeitos da congelaclo sobre blastocistos bovinos. 1986. Thesis Jaboticabal, SP, Brazil. Fair T, Lonergan P, Dinnyes A, Cottel DC, Hittel P, Ware1 FA, Boland MP. Ultrastructure of bovine blastocysts following cryopreservation: effect of method of blastocyst production. Mol Reprod Dev 2001;58:186-195. Alvarenga FCL. Avaliaclo dos efeitos do congelamento e descongelamento sobre a viabilidade e morfologia de embrioes equinos. 1995. Thesis. Botucatu, SP, Brazil. Esper CR, Barbosa JC. Ultra estrutura comparativa de embriBes bovinos. In Congress0 Brasileiro de Reproduczo Animal, MG Brasil, 1991. Hinkorska-Galcheva V, Petkova D, Koumanov K. Changes in the phospholipid composition and phospholipid asymmetry of ram sperm plasma membrane cryopreservation. Cryobiology 1989;26:70-75. Rall WF, Fahy GM. Vitrification: A new approach to embryo cryopreservation. Theriogenology 1985;23:220. Zanenga CA. Freezing on zebu embryos - Development and viability. In: X Congress0 Brasileiro de Reproducgo Animal, 1993 Belo Horizonte, MG, Brazil. Dias AJB, Benchimol M, Fontes RS, Bern AR, Cruz GM. Ultrastructure of frozen bovine blastocysts with glycerol or ethilene glycol . Arq Fat Vet UFRGS, 1997;25(Suppl.):2 15. Rall WF, Wood MJ, Kirby C, Whittingham DG. Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 1987;80:499-504. Rodrigues JL. Aspectos da congelacso de embrioes bovinos. Ars Veterinaria 1992;8:5579.
CRYOPRESERVATION OF BOS TAURUS VS BOS INDICUS EMBRYOS: ARE THEY REALLY DIFFERENT? J.A. Visintin’, J.F.P. Martins’, E.M. Bevilacqua*, M.R.B. Mello’, A.C. Nicacio’, M.E.O.A. AssumpcZo’ ‘Department of Animal Reproduction, Faculty of Veterinary Medicine, *Department of Histology and Embryology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, 05508-000, Brazil ABSTRACT Cryopreservation with storage at very low temperatures is essential to make full use of this technology for both biological and commercial reasons. However, most mammalian cells will die if exposed to these temperatures unless they are exposed to cryoprotectant solutions and cooled and warmed at specific rates. Lowering temperature below 0°C introduces the risk of intracellular ice formation, which likely increases rapidly as the temperature falls. Evidence indicates that ice formation during cooling can cause significantly more damage than ice formation during warming. Comparisons of the toxicity of various cryoprotectants indicated that ethylene glycol (EG) is a nontoxic compound for murine and bovine embryos. The 3.6 M EG solution resulted in similar high survival rates when compared with nonfrozen embryos; deleterious effects of high concentrations of EG became apparent at 7.2 M. The use of EG provides a nontoxic method for the rapid and simplified controlled freezing of in vivo bovine compact morulae-early blastocyst, avoiding the risk of injury caused by high concentrations of cryoprotectants usually required for vitrification. However, in vivo embryos used for freezing and thawing require further studies to understand the ultrastructural changes during the freezing procedure with EG as the single cryoprotectant, especially between Holstein and Nelore cows. This paper describes the ultrastructure of bovine compact morulae-early blastocysts derived by in vivo methods from Holstein and Nelore cows to investigate the fresh morphology as well as that after exposure to cryoprotectant and cryopreservation by conventional slow freezing, quick freezing (nitrogen vapor), and vitrification. Q 2001 by Elsevier Science Inc. INTRODUCTION When cells are frozen, they are subjected to stress resulting from water-solute interactions that arise through ice crystallization. Crystallization induces unfrozen pocket formation of hyperosmotic solution while cooling progresses to approximately -50°C (6). This process results in withdrawal of intracellular water, subsequent cell shrinkage, and possible influx of ions (1, 7). Thawing involves a reversal of these effects, and the consequent inward water flux may cause cell membrane disruption. Because overly rapid freezing causes lethal intracellular ice formation, the optimal cooling rate is thought to be slow enough to prevent this lethal effect, but fast enough to minimize the harmful effects of prolonged exposure to high salt concentrations (“solution effects”). Acknowledgements: The author wishes to acknowledge Dr. Matthew B. Wheeler and Dr. Eric M. Walters for the review in the manuscript. Research was supported by FAPESP. Theriogenology 57345-359. 0 2001 Elsevier Science Inc.
2002
0093-691X/02/$-see front matter PII: SOO93-691X(01)00675-6
Theriogenology
Cryopreservation involves exposure to non-physiologically low temperatures even before freezing occurs. This process is known to induce changes in two-dimensional membrane lipid organization or “packing” (lipid phase transitions) and, in turn, to modify the kinetic properties of intramembranous enzymes (3, 4, 5). Efforts to correlate susceptibility to cryoinjury with the membrane lipid composition among species have suggested that cold shock, a phenomenon by which cryoinjury is induced by sudden cooling without freezing, is more severe when cell membrane sterol concentrations are low and polyunsaturated fatty acid concentrations are high (238). Cryopreserved embryos are widely used in assisted reproduction of domestic animals. Their value in assisted reproduction in cattle is clearly illustrated by the large number of embryos that are frozen and transferred each year. Cryopreservation with storage at very low temperature (e.g., liquid nitrogen at -196’C) is essential to make full use of this technology for both biological and commercial reasons, as it can simultaneously reduce the costs, genetic drift, and disease that are normally associated with maintaining live animals and cell lines. However, most mammalian cells will die if exposed to these temperatures unless they are exposed to cryoprotectant solutions and cooled and warmed at specific rates. Despite considerable difficulties, several effective cryopreservation protocols have been developed for oocytes and embryos of the mouse (13, 22, 26,27) and cow (18,28), and the knowledge gained from this research is starting to be applied to other species. Damage is most likely to occur at temperatures between +15 and -9O’C. Lowering temperature below O’C introduces the risk of intracellular ice formation, which likely increases rapidly as the temperature falls (39). To prevent intracellular ice formation or minimize the damage, all commonly used cryopreservation protocols are designed to dehydrate cells. They usually also achieve very high extra cellular solute concentrations. With regard to slow cooling, this is achieved by placing the cells in a solution containing 10 to 11% (v/v) penetrating cryoprotectant (approximately 1.5 M). The temperature is then lowered, and ice crystal growth is initiated (seeded) within the solution. As the ice crystals grow, the water in the solution is converted from its liquid to a solid state. This increases the concentration of the solutes, which draws water out of the cells. The lower the temperature, the more water can be incorporated into ice, but the rate at which water can leave a cell also falls as the ambient temperature is lowered. The success of slow cooling therefore depends on achieving the optimal balance (equilibrium) between the rate at which water can leave the cell and the rate at which it is converted into ice. Most procedures currently used to cryopreserve embryos, oocytes, and ovarian tissue stipulate a cooling rate of 0.3 to O.S”C/min from the seeding temperature (usually -5 to -9“C) down to a lower temperature, usually between -33 and -4O”C, after which they can be placed in liquid nitrogen. Although cells cooled in this manner still contain some water at the time they are placed in the liquid nitrogen, there is not enough intracellular water to cause damage, provided that appropriate rapid warming protocols are used. Most rapid cooling protocols use solutions with high concentrations of solutes (e.g., cryoprotectants and sugars), which rapidly draw water out of the cells (26,28). In these solutions, cells become sufficiently dehydrated and permeated by cryoprotectants to tolerate direct immersion into liquid nitrogen or nitrogen vapor. These are commonly referred to as nonequilibrium procedures. The non-equilibrium procedures are subdivided into two categories depending on whether or not ice forms in the solution during cryopreservation. A number of solutions are formulated so that they solidify without any ice crystal formation. These are glassforming or “vitrifying” solutions. When measurable or visible amounts of ice form during either
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cooling or warming, the procedures should be noted as rapid cooling (or ultra-rapid cooling) (26). Both the cooling and warming rates, as well as the composition and concentration of the solution will influence how much, if any, ice will form (15,26,28). Evidence indicates that ice formation during cooling can cause significantly more damage than ice formation during warming. Cooling can also disrupt cells in other ways. Oocytes of many, but not all species, are damaged or killed by short-term exposure to temperatures between +15”C and 0°C. This is best documented in pig and cow oocytes and embryos (9, 18, 19,28). The damage may be correlated with the large amount of dark lipid-like material found in these species because lipid removal or lipid polarization reduces chill and cryoinjury (23). Slow cooling procedures can be highly detrimental to oocytes of chill-sensitive species, as they involve prolonged exposure to the temperatures at which most damage occurs, e.g., with the use of a high equilibration temperature and rapid or very rapid cooling rates, good results for oocytes of chill sensitive species can be achieved (12, 17, 19, 28). Strategies to obtain very rapid cooling rates include the use of copper grids, open pulled straws, wire loops, and microdrops on straws sides and foil (16, 19,28). These very rapid cooling rates benefit embryos and oocytes by providing a very fast transit through the temperature zone, which causes damage to embryos and oocytes; it also facilitate vitrification at lower, and hence less toxic, cryoprotectant concentrations. The choice of buffer system, nature of cryoprotectants, and additives such as sugars, calcium chelators, and antioxidants have all been shown to influence cell survival profoundly. It is unlikely that the fundamental role of these substances is to modify the water permeability of the cell membrane; therefore, other explanations for their different and species-species effects must be sought. Cryoprotectant mixtures (e.g., EG and DMSO) may have some advantages over solutions containing only one penetrating cryoprotectant (11,24,28). However, the overall composition of the solution is also important, as embryo and oocyte survival is modified by other components, including sugars, macromolecules, or polymers (10,20,2 1, 22); EGTA (29); and salts (27). High concentrations of polymers such as Ficoll and Dextran (e.g., >30%) appear to be non-toxic to the embryos and can be used to replace an approximately equal amount of ethylene glycol (by weight) without changing the solution’s glass transition temperature (15, 25). It has already been shown (2 1) that low concentrations of Dextran (1 mg/mL) do not benefit oocytes rapidly cooled in 6 M DMSO, but solutions containing only 15% EG (together with 40% Ficoll) allow effective cryopreservation of mouse embryos (14, 15). It now needs to be ascertained whether these polymer-based solutions are suited to oocytes or embryos of other species. The cryoprotectant EG has proved to be nontoxic for murine embryos (4, 36) and may improve the survival rate of frozen and thawed bovine embryos (33, 34, 35, 45). The high penetration rate and the low toxicity of this cryoprotectant allow direct transfer after thawing without preceding dilution (31, 37). Ethylene glycol has been successfully employed at concentrations of 1.5-l .8 M for the freezing of in vivo bovine embryos (30,3 1,32,37). The protocol employing EG at a concentration of 3.6 M was used for successful cryopreservation of IVP bovine embryos in controlled freezing (35). The rapid freezing (nitrogen vapor) of in vivo mouse embryos (40) has been applied with similar success. The rapid penetration of the embryos by EG seems to avoid preferential cryoinjury of sensitive trophoblasts
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and thus allows high survival rates of IVP-expanded blastocysts. The different survival rates following freezing can be attributed to variation in the type and concentration of employed cryoprotectants. Comparisons of the toxicity of various cryoprotectants indicated that EG is a nontoxic compound for murine (36, 38, 40) and bovine (45) embryos. The 3.6 M EG solution resulted in similar high survival rates when compared with nonfrozen embryos; deleterious effects of high concentrations of EG became apparent at 7.2 M. (35). However, when the concentration of EG is gradually increased, embryos tolerate even a final EG concentration of 7.2 M (38,40,41,42). It cannot be ruled out that the direct dilution of the high EG concentration has been deleterious attributable not to the toxicity of the solution but to extreme osmotic stress, resulting in poor survival rates. It has been shown, however, that bovine IVP embryos tolerate direct dilution of a vitrification solution consisting of 40% EG, 6% polyethylene glycol, and 0.5 M sucrose without a decrease in viability (43). Posthaw direct dilution of 1.5 M EG from early mouse embryos was equally efficient as was the dilution with sucrose (40,44). This suggests that the use of sucrose for cryoprotectant dilution is not required to maintain embryonic developmental capacity. The use of EG provides a nontoxic method for the rapid and simplified controlled freezing of in vivo bovine compact morulae-early blastocyst, avoiding the risk of injury caused by high concentrations of cryoprotectants usually required for vitrification. However, in vivo embryos used for freezing/thawing need further studies to understand the ultrastructural changes during the freezing procedure with EG as the single cryoprotectant, especially between Holstein and Nelore cows. The aim of this study was to describe the ultrastructure of bovine compact morulae-early blastocysts derived by in vivo methods from Holstein and Nelore cows and to investigate the fresh morphology as well as that after exposure to cryoprotectant and cryopreservation by conventional slow freezing, quick freezing (nitrogen vapor), and vitrification. MATERIALS AND METHODS Excellent morulae and early blastocysts from Holstein and Nelore cows were produced by in vivo methods and distributed into three groups 1) fresh, 2) exposed to cryoprotectant, and 3) cryopreserved by conventional slow freezing, quick freezing, and vitritiction groups (Table 1). Table 1. Distribution of Nelore and Holstein embryos in fresh, exposed to cryoprotecnt, and freezing group categories. Embryos _.____ __ .-..IProtocols .._, ____.__-. . . . .-.. . -.. . -. .PBS + 0.4 % BSA Fresh 1.8 MEGa Exposed 3.0 M EG + 0.3 M sucrose 7.2 M EG + 0.3 M sucrose+ 18% Ficoll 1.8 M EG - controlled freezing at -0.5 Umin Frozen 1.8 M EG - controlled freezing at -1.2 C/min 3.0 M EG + 0.3 M sucrose - quick freezing 7.2 M EG + 0.3 M sucrose + 18% Ficoll -vitrification “EG =ethylene glycol.
Holstein ...-.- . Nelore ...- - . 3 3 2 2 3 3 1 3 2 3 3 3 3 3 1 2
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To investigate the toxicity of cryoprotectants, embryos were exposed to three different cryopreservation methods: 1) 1.8 M EG for 10 min; 2) 3 M EG + 0.3 M sucrose for 5 min, and 3) pre-equilibration in 3.6 M EG for 3 min and 7.2 M EG + 0.3 M sucrose + 18% Ficoll (MW 70.000) for 1 min. Subsequently, the embryos were washed three times in PBS + 0.4% BSA and prepared for evaluation by light and transmission electron microscopy (Table 1). To investigate cryoinjury, the embryos were exposed to the cryoprotectant and loaded in straws, which contained three columns (in both extremities with PBS + 0.4% BSA and in the middle embryo + cryoprotectant), and frozen. The columns were as follows. 1) 1.8 M EG. The straws were placed at -7°C into a metanol freezer (Haacke, Germany), and seeding was induced with super-cooled forceps after 5 min of equilibration. The samples were cooled at O.S”C/min or 1.2’C/min to -3 l°C and then plunged into liquid nitrogen. 2) 3 M EG. The straws were placed at nitrogen vapor (-17O’C) for 2 min and plunged into liquid nitrogen. 3) 7.2 M EG. The straws were plunged directly in liquid nitrogen. After storage for 1 wk, the straws were thawed at room temperature (22’C) for 10 set in air and in 25“C water bath for 20 sec. The cryoprotectant was diluted inside the straw for 5 min and, after that, the embryos were washed three times in PBS + 0.4% BSA. After thawing and rehydration, the embryos were prepared for evaluation by light and transmission electron microscopy. Embryos were fixed in 2.5% glutaraldehyde in 0.1 M PBS with 0.05 M sucrose for at least 1 hr, removed from fixative, and washed three times for a total of 15 min in 0.1 M PBS. Individual embryos were embedded in 2% agar and then removed in 3-mm3 blocks. Agarose blocks were postfixed in 2% osmium tetroxide (0~04) in 0.1 M PBS for 1 hr, washed three times in 0.1 M PBS for a total of 15 min, dehydrated through ascending concentrations of alcohol, and embedded in Spurr resin. Resin blocks were solidified at 60°C. Semi-thin sections (2 urn) had been stained with toluidine blue for light microscopy. Ultrathin sections (80 nm) from each blastocyst were collected on copper grids and post-stained with uranyl acetate and examined in a transmission electron microscopy (Table 1).
RESULTS AND DISCUSSION Morphology of Non-Frozen (Fresh) Embryos Fresh embryos (n =6) showed discoidal form delimited by the zona pellucida (ZP). In the compact morulae-early blastocyst, the pellucida zone thickness measured 15 pm and, in both cases, presented homogeneous texture in the internal face and more porous aspect in the external faces. The disposition of blastomeres are similar in Nelore and Holstein embryos. The layer of trophoblastic cells in the blastocysts and the layer of embryonic cells in contact with the ZP in morulae revealed elongated form and adhered cells, reflective of a typical epithelium [Figure l(A and C)]. In the blastocyst, the trophoblastic cells surrounded the blastocoele cavity and the inner cell mass. The cells of inner cell mass presented irregular star-like appearance, maintaining spaces between them but establishing occasional junctions [Figure l(B)].
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This arrangement and disposal of the blastomeres in this phase of the development also were reported by other authors, independent of the bovine breed considered (46 47, 48, 53, 54, 55). In the morulae, between the peripheral cells and those disposed more internally, cavities of varied dimensions could be found [Figure l(A and B)]. This finding probably relates to the process of liquid accumulation after the compactation phase and subsequent organization of blastocoele, which increases with embryonic development. Pedersen and Spindle (49), using mouse embryos, demonstrated that the accumulation of fluid in blastocoele is essential to blastomere differentiation. In fact, the cells more peripheral (those that surround these cavities) become more flattened, electrondense, and have an organele content different from the others, indicating that the peripheral blastomere differentiation in trophoblastic cells has been taken. Most of the blastomeres’ nuclei are central, presenting a predominance of eucromatine, one or two proeminent nucleolus. In the blastocysts, the trophoblastic cells revealed a match between themselves by GAP junctions, consisting mainly of tight junctions and desmosomes, which was also observed by other authors (56). Interdigital projections were also observed between the membranes of contiguous cells [Figures l(B and D)]. In the free surface, these cells showed a great amount of microvilus and vesicles covered in the apical region, which indicates an extensive contact surface with the perivitelline space (PvS) specialized in the molecule captation [Figure l(F)]. In this place, cellular debris is frequently found [Figure l(D and F)]. Cells with degenerative features and cellular death were observed similarly in the ICM and in the layer of trophoblastic cells [Figure l(B)]. Other authors had similar findings in bovine (54), mouse (50) equine (56), and monkey (51); those authors categorized these finding as important events for normal embryo development. Enders et al. (52) suggested that, during embryonic differentiation, there is a regulation in the number of blastomeres. This regulation is based on the capacity of the embryo to remove cells that exhibit reduction in its potential development. The ultra-structural morphology and distribution of blastomere cellular compounds showed similarity in the embryos among breeds, which corroborate with the descriptions carried through Crosier et al. (48) and Fair et al. (55). Great numbers of ribosomes and poliribosomes prevailed in the cytoplasm of these cells, as compared with other organeles. Differences, however, had been observed in the pattern and concentration of the lipid droplets, similar to endosomics, phagocytics vacuoles, and mitochondria structures (Table 2). The lipid droplets were larger and in greater number in the Holstein embryos. Vesicles of variable dimensions with contents of different electrondensities that presented partially digested material were found in the blastomeres, showing a predominance of this phenomena in Nelore [Figure l(E)]. These structures appear to form fagosomic vacuoles with or without the secondary fusing of lisosomes. Rounded and prolongated mitochondria with transversal crystals were more abundant in the cells of Nelore embryos. Differences in these cellular components between Bos taurus taurus and Bos taurus indicus embryos had also been found by Esper and Barbosa (57); however, little literature of regarding this is well known. Table 2. Morphologic differences among Nelore and Holstein embryos.a Holstein Characteristics ---. .^.-._..___ . Nelore -----._._..._--..--..-_._.-. ++ + Mitochondria ++ +++ Lipid droplets +++ ++ Endosomic cistern vesicles “The values had been estimated on the basis of morphologic analysis: + (few), ++ (intermediate values), and +++ (many).
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Morphology of Exposed Embryos to the Cryoprotectants The protocols of cryopreservation in both breeds revealed low toxicity to the embryos that, morphologically, presented similar features compared with fresh embryos. The few differences observed in relation to cryopreservation refer to a larger PvS where the distance between ZP and blastomeres increases [Figure 2(A)]. Eventually, deformities in relation to the original form of the blastocysts were observed [Table 3 and Figure 2(B)]. According to Dias et al. (61) and Alvarenga (56), who analyzed bovine embryos, the increase of PvS refers to the incomplete re-expansion of the blastomeres during the rehydratation of the cells, which is associated with cryoprotectant removal. Table 3. Alterations caused by exposure of Nelore and Holstein embryos to the cryoprotectants.a Alterations observed after cryoprotectant exposure ..- - - . --_.- . --__-.-.. .- ._._ -... ..___.._.._ . . .-....Increase of the perivitelline space Alterations in the embryo form “(+) present / (-) not present.
Nelore and Holstein Embryos ~. . . _..-._... ... -. .-_ .___. + +
Morphology of Frozen Embryos Although after thawing, the frozen embryos apparently kept their form and similar structure in comparison with the fresh embryos, a detailed analysis showed that the embryonic cells, mainly in Nelore, presented significant alterations. Independent of the protocol used for freezing and the origin of the embryo, embryonic cells presented qualitative and quantitative signs of degeneration and cellular death when compared with fresh embryos (Figure 3). These cells occupied the same areas as cells with normal appearance but showed mitose signs. One of the most common degenerative features was the swelling of cells with cytoplasmatic injuries and vacuolization nucleous [Figure 3(A and B)]. Cellular lysis was also observed, as well as cells with discontinuous plasmatic membranes [Figure 3(A)]. These findings are similar to those observed during the processes of cellular death at necrosis or in cells submitted to hyperosmotic stress, which are characteristic of alterations in permeability of membranes. Similar comments were also been reported by Alvarenga (56) when equine embryos were frozen. The author describes the process in terms of alterations in the distribution of lipids and proteins in the membrane, causing abnormal rearrangements of these molecules and subsequent changes in the cellular metabolism. Hinkorska-Galcheva (58) affirmed that modifications in the molecular organization of lipids in the membranes can cause structural and functional alterations of its enzymes and proteins. It is possible that our findings also reflect the deleterious action of the freezing and thawing process among blastomeres through alterations in permeability of plasmatic and organelle membranes. The presence of cellular debris was also observed in blastocoele; PvS; and, in some samples, in the intercellular space. The presence of great intra-embryonic cavities [Figure 3(A and C)] was also observed. Possibly, this cellular debris precedes the lysis process following the cryopreservation process. Morphologic features of the embryonic cells of Nelore and Holstein embryos were better preserved under control freezing than under quick freezing and the vitrification process. However, during the quick freezing and vitrification processes, Holstein embryos exhibited better morphologic conditions than did Nelore embryos. These results confirm
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the results of Zanenga (60) who showed that homogeneous behavior during the freezing process for Bos tam-us embryos, but not Bos indicus embryos, varied in quality after thawing, resulting in lower pregnancy rates. However, studies carried out by Rail, Fahy (59), and Rall et al. (62) demonstrated the viability of mouse embryos after devitrification (thawing). Rodrigues (63) related that vitrification results in bovine are unsatisfactory. Other significant findings were the sensible reduction of microvilus on the surface of the trophoblastic cells [Figure 3(E)] and an increase in the incidence of vesicles of the endosomic system, mitochondrial vacuolization [Figure 3(D)], and intense expansion of membrane compartment (endoplasmatic reticulum and/or Golgi apparatus).
Table 4. Alterations caused by different freezing processes on Nelore and Holstein embryos.a
Freezing protocols
-0.5’C
Nelore -1.2’C Vapor7%%s
-0.5’C
Holstein -1.2’C Vapor
Vitrif
Dead cells
++
++
+++
+++
+
+
++
++
Lysed cells
++
++
+++
++
+
+
++
++
Swollen cells
+++
+++
+++
+++
++
++
+++
+
Cellular debris
+
+
+++
+++
++
++
++
++
++
+
+
+++
+++
+++
++
+++
+++
++
++
++
++ ++ ++ ++ + Microvilus on trophoblastic surface +++ ++t +++ +++ +++ Vesicle of endossomic system ++ +++ +++ ++ ++ Mitochondrial vacuolization +++ + + + +++ Cistern enlargement of REG and Golgi aThe values had been estimated on the basis of morphologic analysis: + values), and +++ (many). bVitrif = vitrification.
(few), ++ (intermediate
Theriogenology
353
Figure t. Fresh embryos. A) Holstein embryo (zp = zona pellueida, tf = differentiation of trophoblastic cell, icm = inner cell mass, and * = cavities between icm and tf); LM × 1000. B ) N e l o r e embryo. Contiguous inner cells mass (de = degenerated cell arrows, interdigital projections); TEM x 3000. C) Nelore embryo; L M × 1000. D) Holstein embryo. Cytoplasmic detail ofthrophoblastic cell (pvs = perivitelline space, arrow = interdigital projection, black arrow = cellular debris in pvs, and * = large lipid droplets); TEM × 4000. E) Nelore embryo. Cytoplasmic detail ofthrophoblastic cell (N = nucleous; * = vesicles presenting a partially digested material); TEM × 10000. F) Holstein embryo. (arrow = microvilli in trophoblastic call surface, black arrow = cellular debris in pvs, and * = some lipid droplets); TEM x 2000.
354
Theriogenology
Figure 2. Embryos after cryoprotectant exposure. A) Nelore embryo showing cellular aspects; TEM x 1000. a) Insert of Nelore embryo after cryoprotectant exposure showing larger perivitelline space; LM × 400. B) Holstein embryo showing cellular aspects; TEM × 2000. b) Insert of Holstein embryo deformed after cryoprotectant exposure; LM × 400.
Theriogenology
355
Figure. 3. Frozen and thawed embryos. A) Nelore embryo with various cytoplasmatic injuries (zp = zona pellucida, pvs = perivitelline space, arrow = cellular debris in PvS, black arrow = cellular lysis, * = generated vacuolization, and + -- nuclear vacuolization); T E M x 1500. a) Insert o f Nelore embryo showing a similar structure to the fresh one; LM x 400. B) Holstein embryo with various cytoplasmatic injuries (N = nucleous, arrow - nuclear injuries, and * = cytoplasmatic vacuolization); T E M x 2000. b) Insert o f Holstein embryo showing a similar structure to the fresh one; LM x 400. C) Holstein embryo (N = some nucleous; * = great intra-embryonic cavities); T E M x 1200. D) Holstein embryo (arrow = mitochondrial vacuolization; * = intense expansion o f membranous compartment); T E M × 7500. E) Nelore embryo (N = nucleous; arrow = absence o f microvilli on surface of trophoblastic cell); T E M x 1500.
Theriogenology
In conclusion, Holstein embryos exhibit more intracellular lipid when compared with Nelore embryos. Embryos exposed to cryoprotectants demonstrated none or few morphological alterations in both breeds, indicating that embryonic cells were mildly affected by cryoprotectant toxicity. Holstein and Nelore embryos frozen by the slow freezing method had better morphological quality than quick freezing and vitrification. In the quick freezing and vitrification REFERENCES 1.
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