The association of resveratrol and AFPI did not enhance the cryoresistance of ram sperm.

Cryoprotectants are required to reduce damage caused to the cells due to low temperatures during the cryopreservation. Antifreeze proteins (AFP) have a well-known role in cell membrane protection, while resveratrol is a potent antioxidant. This study assessed the effect of the association of resveratrol concentrations and AFP I in a ram semen extender. Pooled semen of four rams was allocated into six treatments in a factorial arrangement: (CONT, only the semen extender); only AFP I (ANT: 0.1 µg/mL of AFP I), only resveratrol, one treatment with two levels (10 µM/mL or 50 µM/mL of resveratrol); and two treatments with the interactions, with one AFP I and one of the two levels of resveratrol (0.1 µg/mL of AFP I with 10 µM/mL resveratrol; 0.1 µg/mL of AFP I with 50 µM/mL resveratrol). No interaction between factors was observed on sperm kinetics, plasma membrane integrity, hypo-osmotic test, and mitochondrial activity parameters. There was a high probability (P = 0.06) of reducing sperm cells with functional membrane percentage in the hypo-osmotic test and increasing the percentage of sperm with high mitochondrial activity (P = 0.07) was observed in AFP presence. An interaction of AFP and resveratrol was observed in non-capacitated sperm (P = 0.009), acrosomal reaction (P = 0.034), and sperm binding (P = 0.04). In conclusion, the association of resveratrol and AFP did not improve the quality of frozen-thawed semen and even promoted deleterious effects compared to their single addition in the semen extender. The supplementation of 50 µM/mL of resveratrol improved the outcomes of frozen-thawed ram sperm, being a potential cryoprotectant.

Slow Freezing of Preserved Boar Sperm: Comparison of Conventional and Automated Techniques on Post-Thaw Functional Quality by a New Combination of Sperm Function Tests.

The slow freezing of boar sperm is the only way to preserve genetic material for extended periods; this can be achieved with exposure to liquid nitrogen vapors (conventional) or by using automated freezing equipment. The aim was to compare the effect of both techniques on post-thaw functionality. Boar sperm devoid of seminal plasma and resuspended in lactose-egg yolk-glycerol medium were cryopreserved. Conventional: straws were exposed to LN2 vapors; automated: using a drop curve of -39.82 °C·min-1 for 113 s from -5 to -80 °C during the critical period; and subsequent immersion in NL2. Cell viability, cholesterol flow, mitochondrial membrane potential (MMP), lipid peroxidation, peroxynitrite, superoxide anion levels, phosphatidylserine translocation, and caspase activation were evaluated by flow cytometry. In addition, total motility (TM) and progressive motility (PM) were determined by the SCA system immediately (T0), 60 (T60), and 120 min (T120) post-thawing. Automated freezing significantly reduces cholesterol flow and free radical and lipid peroxidation levels, making it possible to preserve motility for 120 min of incubation. At the same time, viability, acrosome integrity, MMP, and caspase activation did not differ from the conventional technique. In conclusion, controlling the temperature drop curve using automated freezing equipment reduces oxidative/nitrosative stress, preserving membrane fluidity and sperm motility.

The initial steps toward the formation of somatic tissue banks and cell cultures derived from captive Antillean manatee (Trichechus manatus manatus) skin biopsies.

The declining population of the Antillean manatee caused by ecosystem degradation and rising pollution has prompted interest in developing conservation strategies for this species. Given this scenario, somatic tissue banks are important tools for acquiring knowledge about the species, as well as for obtaining somatic cells for biotechnological and ecotoxicological applications. Therefore, we aimed to assess the effects of slow freezing (SF) and solid-surface vitrification (SSV) of the dermis of captive Antillean manatees on the histology and ultrastructure of the tissue and cell viability in culture. While the SSV did not change the dermis thickness, the SF maintained the tissue proliferative potential, assessed by the nucleolar organizer region area, similar to noncryopreserved tissues. Moreover, both techniques reduced the number of fibroblasts and increased the percentage of collagen fibers. Nevertheless, only tissues cryopreserved with SF and noncryopreserved tissues were able to produce cells after in vitro culture. Although SF did not alter cell viability and proliferative activity, cells derived from cryopreserved tissues showed decreased metabolism, altered apoptosis, increased levels of reactive oxygen species, and mitochondrial membrane potential compared to cells from noncryopreserved tissues. In summary, we demonstrated for the first time that Antillean manatee somatic tissues can be cryopreserved by SF, and cells can be obtained after in vitro culture. Improvements in cryopreservation conditions, especially vitrification, of somatic samples are needed to increase the quality of somatic tissue banks in this species.

Definition of protocols for cryopreservation and three-dimensional in vitro culture of prepubertal goat testicular tissue after histomorphological, ultrastructural, and functional analysis.

This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution - ES: 10% DMSO and 10% ethylene glycol - EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm3 showed a higher percentage than fragments of 9 mm3. Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation.

Effect of the addition of antifreeze protein type I on the quality of post-thawed domestic cat epididymal sperm.

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen-thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 µg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

Delivering embryos following 10 years of cryopreservation, using unpaired freeze/thaw techniques: A case report.

Although frozen embryo transfer is a widely established route for assisted reproduction, successful frozen embryo transfer using embryos that have undergone long term cryopreservation remains relatively unexplored, and its efficacy remains a matter of some debate. This case report describes two successful frozen embryo transfer conceptions in the same patient, one after 3 months of cryopreservation and the second 10 years after cryopreservation. These embryos were cryopreserved using the slow freezing technique and were thawed using an unpaired technique (ultra-rapid warming) after 10 years of storage.

Establishment, characterization, and cryopreservation of cell lines derived from red-rumped agouti (Dasyprocta leporina Linnaeus, 1758) - A study in a wild rodent.

Somatic cells can be used for rescuing wild mammals of ecological and economic importance, such as red-rumped agouti, through their application in advanced technologies. Thus, appropriate cell isolation, culture, and storage through cryopreservation can ensure the future safe use of these cells. We aimed to establish and evaluate the effects of culture time (second, fifth, and eighth passages) and cryopreservation on the morphology, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis on somatic cells derived from red-rumped agouti skin. Initially, we identified six dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the second, fifth, and eighth passages, as well as the cryopreservation conditions used did not affect the metabolism or level of apoptosis. Nevertheless, cells in the fifth passage featured a reduction in proliferative activity and an increase in ROS levels when compared to second and eighth passage cells. Moreover, cryopreservation resulted in reduced ΔΨm when compared to non-cryopreserved cells. Additionally, cryopreserved cells showed a reduction in viability immediately after thawing; nevertheless, the viability of these cells was re-established after 11 days of in vitro culture and was similar to that of non-cryopreserved cells. In conclusion, we have shown that viable fibroblasts can be obtained from red-rumped agouti skin, featuring minimal changes after eight passages in in vitro culture systems. Additionally, adjustments to the cryopreservation protocol are necessary to reduce cellular oxidative stress caused by low temperatures.

Comparison between two cryopreservation techniques of human ovarian cortex: morphological aspects and the heat shock response (HSR).

This study was tailored to compare the cryopreservation of the human ovarian cortex using closed metal container vitrification or the slow-freezing technique. Superficial ovarian cortical tissue biopsies were collected from 12 participants who underwent gynaecological videolaparoscopy. The fragmented samples were allocated to three experimental conditions: (a) fresh ovarian tissue, (b) slow-freezing, and (c) vitrification with a metal closed container. After thawing or rewarming, cellular morphological analyses were performed to determine tissue viability. The cellular response to thermal stress was measured by a putative increase in the immune quantification of the heat shock protein 70 kDa (heat shock protein 70 kDa response - HSR) after a heat challenge (2 h exposure at 42 °C). Both the total number of intact follicles and the frequency of primordial follicles were higher in fresh ovarian tissue than in the preserved samples, regardless of the technique employed. There was a trend towards an increase in the absolute number of intact follicles in the tissue preserved by vitrification. After cryopreservation, a higher HSR was obtained after slow-freezing. These results indicate that both cryopreservation techniques present advantages and may be used as alternatives to ovarian tissue cryopreservation.

Investigation on revascularization time and initial damage after transplantation of fresh and cryopreserved ovarian tissue in domestic cats.

The present study evaluated revascularization time of fresh and cryopreserved cat ovarian tissue after transplantation to subcutaneous tissue. Ovaries of five cats were used and eight pieces of ovarian tissue were taken from each pair of ovaries. Immediately after removal, three pieces were transplanted and one fixed for fresh control. The remaining four pieces were cryopreserved and, after thawing, one was fixed for cryopreservation control and three were transplanted. Grafts were recovered on days 2 (D2), 4 (D4) and 6 (D6) post-transplantation. Blood vessels were identified by immunohistochemistry and doppler ultrasound. Immunohistochemistry showed that the percentages of total tissue area occupied by blood vessels were similar (P > 0.05) in fresh and cryopreserved tissues. In both cases, blood vessel area was significantly higher (P < 0.05) on D4 and D6 compared to D0. Ultrasound analysis showed vascularization improvement on the periphery of grafts from D2 to D4 and from D4 to D6, both in fresh and cryopreserved tissue samples. Nonetheless, there was a significant decrease (P < 0.05) in the percentage of morphologically normal follicles (MNF) after transplantation compared to non-transplanted tissue (D0), both for fresh and cryopreserved samples. Moreover, the number of follicles found in samples was considerably smaller after grafting. In conclusion, revascularization of ovarian tissue autotransplanted to subcutaneous tissue in domestic cats occurs within 4 days after transplantation, both for fresh and cryopreserved tissue. However, large follicular loss has been observed in the first days post-transplantation, especially in cryopreserved tissues.

Recent progress in bovine in vitro-derived embryo cryotolerance: Impact of in vitro culture systems, advances in cryopreservation and future considerations.

Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.

Taxas de maturação de oócitos congelados lentamente de caprinos / Slowly frozen caprine oocyte maturation rate

The objective was to evaluate the maturation rates of goat oocytes submitted to slow freezing in conventional medium for IVM. For this purpose, cumulus-oocyte complexes aspirated from pubic goat ovaries were classified morphologically and slowly frozen with 1.5 M ethylene glycol. After thawing the cryopreserved oocytes, those classified as viable were matured in conventional in vitro maturation medium. Evaluating the maturation rate, the percentage of matured oocytes in the group that underwent the cryopreservation process is significantly lower (17.6%) when compared to the control group (69.2%), also showing a high percentage of immature oocytes. Several oocyte injuries were found, caused by the studied cryopreservation method, interfering with their oocyte competence. Even with nuclear maturation rates, observed through the extrusion of the first polar corpuscle, the morphologies were altered in most oocytes, and further studies using new techniques and / or other cryoprotectants are necessary.(AU)

Taxas de maturação de oócitos congelados lentamente de caprinos / Slowly frozen caprine oocyte maturation rate

The objective was to evaluate the maturation rates of goat oocytes submitted to slow freezing in conventional medium for IVM. For this purpose, cumulus-oocyte complexes aspirated from pubic goat ovaries were classified morphologically and slowly frozen with 1.5 M ethylene glycol. After thawing the cryopreserved oocytes, those classified as viable were matured in conventional in vitro maturation medium. Evaluating the maturation rate, the percentage of matured oocytes in the group that underwent the cryopreservation process is significantly lower (17.6%) when compared to the control group (69.2%), also showing a high percentage of immature oocytes. Several oocyte injuries were found, caused by the studied cryopreservation method, interfering with their oocyte competence. Even with nuclear maturation rates, observed through the extrusion of the first polar corpuscle, the morphologies were altered in most oocytes, and further studies using new techniques and / or other cryoprotectants are necessary.

Comparative analysis between slow freezing and ultra-rapid freezing for human sperm cryopreservation.

OBJECTIVE: Cryopreservation of human spermatozoa is fundamental in assisted reproductive technology. At present, slow freezing techniques are widely used for sperm cryopreservation. Recently, sperm vitrification has been proposed as an alternative to slow freezing. This study aimed to compare the efficiency of slow versus ultra-rapid freezing after thawing and to determine the level of DNA fragmentation in post-thaw normal human semen samples processed through each of the cryopreservation techniques. METHODS: Ultra-rapid freezing is a method that only differs from conventional ultra-rapid freezing in the use of sucrose as a cryoprotectant. In experiment 1, 24 semen samples were used to compare sperm recovery rates after slow and ultra-rapid sperm freezing. In experiment 2, 18 semen samples were used to compare post-thaw sperm DNA fragmentation levels after each of the cryopreservation techniques. RESULTS: In experiment 1, no significant differences were observed in sperm concentration recovery rates, although slow freezing showed a lower progressive motility rate than ultra-rapid freezing (16.6±7.4% vs. 34.7±10.2%), and higher non-progressive and immotile sperm counts (9.0±4.0% vs. 7.6±2.8%; and 74.4±10.1% vs. 57.8±10.3%, respectively). In experiment 2, sperm DNA fragmentation after thawing was significantly higher in slow freezing than in fresh post gradient processing and ultra-rapid freezing samples (47.3±13.4% vs. 9.1±3.7% vs. 14.6±4.6%, respectively). CONCLUSION: Sperm ultra-rapid freezing may be an alternative to slow freezing with better recovery results and less apparent DNA damage.

Cryopreservation and characterization of canine preantral follicles.

The aim of this study was to define the population, morphological and ultrastructural characteristics of bitch preantral follicles (PAFs) and to compare the effects on the morphology of PAF of two cryopreservation techniques - slow freezing (SF) and vitrification (V) - of bitches' ovarian tissue. The average population (number per ovary) of PAFs was 48,541 ±â€¯18,366, where 94.25% were primordial (45,145 ±â€¯16,076). The average diameter of the primordial follicles was 27.5 ±â€¯4.2 µm. The overall percentage of morphologically normal PAFs was 93.66 ±â€¯6.81% for the control group, 86.16 ±â€¯11.05% after SF and 68.14 ±â€¯12.75% after V. The percentage of normal primordial follicles was 96.69 ±â€¯4.72% in control, 89.51 ±â€¯10.39% in SF and 75.32 ±â€¯9.23% in V. There was no significant difference in the overall percentage of normal PAFs among SF and the control. However, slow frozen follicles presented ultrastructural damage, while vitrified primordial and primary follicles were well preserved. In conclusion, although slow freezing seemed to be a good preserving method, vitrification was more effective than slow freezing in preserving the ultrastructure of primordial and primary follicles of bitches.

Freezing goat embryos at different developmental stages and quality using ethylene glycol and a slow cooling rate / Congelação de embriões caprinos em diferentes estádios de desenvolvimento e qualidade utilizando etileno glicol e taxa de resfriamento lenta

The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20ºC to -6ºC at a cooling rate of 3ºC/min, stabilization for 15min (seeding after 5min), from -6 C to -32ºC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37ºC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37ºC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos.(AU)

Este estudo testou a eficiência de protocolo alternativo de criopreservação de embriões caprinos de diferentes qualidades morfológicas. Foram utilizados 58 embriões, coletados entre o sexto e o sétimo dia do ciclo estral (n=29/grupo). Embriões congelados passaram por solução 1,5M etilenoglicol por 10min e foram aspirados durante esse tempo para parte central de palheta 0,25mL, separada por bolhas de ar de colunas contendo PBS 0,4% BSA e 20% SFB. As palhetas foram congeladas em máquina de congelação de 20ºC a -6ºC, com taxa de resfriamento de 3ºC/min, estabilização por 15min (seeding após 5min), -6ºC a -32ºC a 0,6ºC/min, e imersas em nitrogênio líquido. Os embriões foram descongelados por 30s a 37ºC, em água. Embriões frescos foram mantidos em solução de manutenção (37ºC). Embriões frescos e congelados/descongelados foram transferidos para 30 receptoras no sétimo dia do ciclo estral. A taxa de partos e a de crias nascidas (respectivamente) foram similares (P>0,05) para receptoras recebendo embriões frescos (66,7% ou 10/15; 55,2% ou 16/29) ou congelados/descongelados (60,0% ou 9/15; 51,7% ou 15/29). O protocolo de criopreservação de embriões utilizado no presente estudo resultou em índices de eficiência semelhantes aos de embriões frescos.(AU)

Freezing goat embryos at different developmental stages and quality using ethylene glycol and a slow cooling rate / Congelação de embriões caprinos em diferentes estádios de desenvolvimento e qualidade utilizando etileno glicol e taxa de resfriamento lenta

The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20ºC to -6ºC at a cooling rate of 3ºC/min, stabilization for 15min (seeding after 5min), from -6 C to -32ºC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37ºC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37ºC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos.(AU)

Este estudo testou a eficiência de protocolo alternativo de criopreservação de embriões caprinos de diferentes qualidades morfológicas. Foram utilizados 58 embriões, coletados entre o sexto e o sétimo dia do ciclo estral (n=29/grupo). Embriões congelados passaram por solução 1,5M etilenoglicol por 10min e foram aspirados durante esse tempo para parte central de palheta 0,25mL, separada por bolhas de ar de colunas contendo PBS 0,4% BSA e 20% SFB. As palhetas foram congeladas em máquina de congelação de 20ºC a -6ºC, com taxa de resfriamento de 3ºC/min, estabilização por 15min (seeding após 5min), -6ºC a -32ºC a 0,6ºC/min, e imersas em nitrogênio líquido. Os embriões foram descongelados por 30s a 37ºC, em água. Embriões frescos foram mantidos em solução de manutenção (37ºC). Embriões frescos e congelados/descongelados foram transferidos para 30 receptoras no sétimo dia do ciclo estral. A taxa de partos e a de crias nascidas (respectivamente) foram similares (P>0,05) para receptoras recebendo embriões frescos (66,7% ou 10/15; 55,2% ou 16/29) ou congelados/descongelados (60,0% ou 9/15; 51,7% ou 15/29). O protocolo de criopreservação de embriões utilizado no presente estudo resultou em índices de eficiência semelhantes aos de embriões frescos.(AU)

Comparison between Slow Freezing and Vitrification in Terms of Ovarian Tissue Viability in a Bovine Model / Comparação da viabilidade do tecido ovariano após congelamento lento e vitrificação em modelo bovino

Abstract Objective To assess the viability of bovine ovarian tissue after cryopreservation through either slow freezing or vitrification, and to compare it to that of control tissue by performing morphological analyses. Methods The study included 20 bovine ovarian cortex fragments that were divided into control, vitrification, and slow freezing groups. Each group consisted of four fragments of the same ovary, two fixed without cultivation, and two fixed with cultivation. Tissues were evaluated based on follicular morphology immediately after heating and after 7 days of culture, and compared with the control group. Results A total of 240 fragments were analyzed, generating a sample of 1,344 follicles without cultivation and 552 with cultivation. When the non-cultivated samples were classified as non-atretic follicles, 572 were found in the control group, 289 in the vitrification group, and 373 in the slow freezing group, showing no significant differences. When classified as atretic, 46 follicles were found in the control group, 23 in the vitrification group, and 41 in the slow freezing group, also showing no statistical difference. In the post-culture sample, an evolution of the follicular stages could be observed. This finding was important to support that the follicles considered non-atretic in the non-cultivated group were actually viable in the morphological evaluation. Conclusion With no differences between the protocols, vitrification was shown to be an advanced and alternative method for patients who will undergo treatments that.

Resumo Objetivo avaliar a viabilidade do tecido ovariano bovino após a criopreservação, utilizando congelamento lento e vitrificação, e comparando com o tecido controle por meio de análises morfológicas. Métodos o estudo incluiufragmentos de córtex de vinte ovários bovinos divididos em grupos controle, vitrificação e congelamento lento. Cada grupo foi composto por quatro fragmentos do mesmo ovário, sendo dois fragmentos fixados sem cultivo e dois fragmentos fixados pós-cultivo. Os tecidos foram avaliados pela morfologia folicular logo após o aquecimento e após sete dias de cultivo, e comparados com o grupo controle. Resultados um total de 240 fragmentos foi analisado, gerando uma amostra de 1.344 folículos sem cultivo e 552 pós-cultivo. Quando a amostra sem cultivo teve seus folículos agrupados em não atrésicos, obtivemos 572 no grupo controle, 289 no vitrificação, e 373 no congelamento lento, não apresentando diferença estatística. Quando agrupados em atrésicos, o grupo controle apresentou 46 folículos, o vitrificação, 23, e o congelamento lento, 41, não apresentando também diferença estatística. Na amostra pós-cultivo, podemos observar uma evolução dos estágios foliculares: esse achado foi importante para sustentar que os folículos considerados não atrésicos na avaliação morfológica sem cultivo estavam realmente viáveis. Conclusão não havendo diferenças entre os protocolos, a vitrificação se mostra um avanço e um método alternativo para pacientes que irão se submeter a tratamentos que podem levar a uma falência ovariana, uma vez que a metodologia é mais barata, mais rápida e mais bem adaptável a uma rotina de um laboratório.

What is new in the cryopreservation of embryos?

Embryo cryopreservation represents a pivotal tool for the long-term storage and exchange of valuable genetic resources of livestock and endangered species. The innumerable applications of embryo cryopreservation in human medicine, animal production, as well as in other embryo biotechnologies for research purposes are calling for standardized protocols that can be used in these different fields. This review will provide the reader with a brief outline to “the classics” of embryo cryopreservation procedures in farm animals and with a deeper insight into “the new trends”. Moreover, the cryopreservation effects on the embryo will be revised; from the easily visible cellular damage to the damage at the transcriptomic, proteomic and lipidomic level, and fresh attention will be given to the epigenetic effects of this technology. Finally, we will go through personal considerations to take into account when embryo cyopreservation is used: how to select the best embryos for cryopreservation, the eternal question for how long can we store the cryopreserved embryos, the fact that size and fat matter when it comes to embryo cryopreservation and ultimately, our suggestion about designing cryopreservation protocols “à la carte ” attending the needs of each type of embryo.(AU)

What is new in the cryopreservation of embryos?

Embryo cryopreservation represents a pivotal tool for the long-term storage and exchange of valuable genetic resources of livestock and endangered species. The innumerable applications of embryo cryopreservation in human medicine, animal production, as well as in other embryo biotechnologies for research purposes are calling for standardized protocols that can be used in these different fields. This review will provide the reader with a brief outline to “the classics” of embryo cryopreservation procedures in farm animals and with a deeper insight into “the new trends”. Moreover, the cryopreservation effects on the embryo will be revised; from the easily visible cellular damage to the damage at the transcriptomic, proteomic and lipidomic level, and fresh attention will be given to the epigenetic effects of this technology. Finally, we will go through personal considerations to take into account when embryo cyopreservation is used: how to select the best embryos for cryopreservation, the eternal question for how long can we store the cryopreserved embryos, the fact that size and fat matter when it comes to embryo cryopreservation and ultimately, our suggestion about designing cryopreservation protocols “à la carte ” attending the needs of each type of embryo.

Comparison of vitrification and conventional freezing for cryopreservation of caprine embryos.

The experiment aimed to compare conventional freezing and different vitrification protocols for cryopreservation of caprine embryos at morphological, ultrastructural, and functional levels. Caprine embryos produced in vivo were allocated randomly to three groups: (1) conventional freezing with ethylene glycol (EG); (2) dimethyl sulfoxide + EG (DMSO/EG) vitrification; and (3) dimethylformamide + EG (DMF/EG) vitrification. All groups were scored for cell viability (propidium iodide staining and ultrastructural levels) and re-expansion rate after thawing or warming. Embryos subjected to DMSO/EG vitrification showed higher cell viability (73.33%), compared with DMF/EG vitrification and conventional freezing group embryos (40.00 and 66.66%, respectively). The ultrastructural study revealed that vitrified embryos had greater preservation of cellular structure than embryos from conventional freezing with EG. DMSO/EG vitrification resulted in higher rates of re-expansion in vitro (47.36%) than DMF/EG vitrification (31.58%), and conventional freezing (25.00%). In conclusion, caprine embryos produced in vivo are better cryopreserved after vitrification than conventional freezing, therefore we conclude that DMSO/EG vitrification is the most effective protocol for cryopreservation.