The cryopreservation of mammalian embryos has become an integral part of method s to control animal reproduction. Numerous vitrification solutions have been formulated with ethylene glycol in combination with macromolecules, sugars and other cryoprotective agents. These indicate that a study of ethylene glycol as a cryoprotectant of choice in vitrification studies would be promising. To understand the cryobiology of ethylene glycol, several factors have to be studied. These are : cryoprotectant toxicity, osmotic stress and temperature at exposure. Understanding these factors could lead to the formulation of vitrification protocols that would lead to higher viability rates after cooling. First, ethylene glycol must be used as the sole cryoprotectant in a solution without macromolecules and sugars. Second, partial dehydration and permeation prior to cooling to subzero temperatures must be studied to achieve accurate exposure and a one-step dilution method. Third, the toxic effects of ethylene glycol must be overcome without sacrificing its vitrification properties by combining step-wise exposure at appropriate temperatures, low concentration and decreased volume. Fourth, the long-term effects of ethylene glycol on exposed or vitrified embryos must be determined. Lastly, the influence of culture on the viability of vitrified embryos must be studied to improve viability rates after warming

Ovulated mouse oocytes denuded of their cumulus cells, were vitrified in a solution containing 7 M ethylene glycol as the sole cryoprotectant using one or two steps of exposure before vitrification and were diluted in 1 M sucrose solution in 5 or 10 min after warming. The results proved that the viability of oocytes are detrimentally affected by exposure to the vitrification solution even without vitrification. At 5 min dilution time, the two-step exposure was superior to the one-step in terms of the post-warming recovery rate of vitrified oocytes with normal morphology and their subsequent development to the blastocyst stage (p0.001) after fertilization in vitro. At 10 min dilution time, no significant difference between one or two-step exposure was found. The effect of the addition of 0.5 M sucrose to the vitrification solution was also determined and did not result in a significant improvement in the viability of oocytes vitrified in one-step and diluted for 10 min. In conclusion, the results in this study indicate that oocytes can be vitrified with 7 M ethylene glycol as the sole cryoprotectant in the vitrification solution, and that the recovery of normal oocytes after one-step exposure in the vitrification solution can be improved by 10 min dilution time. However, the improvement in the recovery rate of oocytes with normal morphology and their subsequent developmental in vitro was not improved by the addition of 0.5 M sucrose to the vitrification solution

A study was made to determine if mouse zygotes can be effectively vitrified in 7 M ethylene glycol in modified Dulbecco's phosphate buffered saline (PB1) and to find out if the development of vitrified-warmed zygotes in vitro can be improved by renewing the culture medium. The results showed that without medium change, vitrification reduced the development of zygotes to the expanded blastocyst stage (p0.01). With medium change, the development rate of vitrified-warmed zygotes exposed in 7 M ethylene glycol for 1 or 2 min was similar to that of unvitrified zygotes. However, prolonged exposure (5 min) markedly reduced the development rates of vitrified-warmed zygotes to the expanded blastocyst stage (p0.05). When the zygotes were vitrified in 7 M ethylene glycol and diluted at 18 degree C to 22 degree C, a slower efflux of ethylene glycol from the cell might have occurred, leading to a toxic effect of ethylene glycol in culture. The development rates of vitrified embryos cultured with medium change at 24 hr did not significantly differ from the untreated control (89.0% vs 96.5%). In conclusion, this study showed that mouse zygotes can be vitrified in 7 M ethylene glycol in PB1 and that changing the culture medium can improve the in vitro development rates of vitrified-warmed zygotes to the expanded blastocyst stage

The use of frozen semen has become increasingly popular in canine artificial insemination, stimulating research to improve freezing methods and extenders. In this study, was compared the final concentration of 5% ethylene glycol used in 3 equilibration protocols. In Method I, one part of semen was added to two parts of tris-fructose-citric acid with 7.5% ethylene glycol (Extender 2) and frozen without cooling. In Method II, one part of semen was added to two parts of tris-fructose-citric acid with 7.5% ethylene glycol (Extender 2) and cooled at 5ºC for 1 hour before freezing. In Method III, one part of semen was added to one part of tris-fructose-citric without ethylene glycol (Extender 1) and cooled at 5ºC for 1 hour. Just after this period, one part of 5ºC cooled tris-fructose-citric acid containing 15% ethylene glycol (Extender 3) was added to the previous extended semen, maintained at 5ºC for one additional hour and frozen. For freezing in each procedure semen was packaged in 0.5 ml plastic straws and placed for 20 minutes in vapor 5 cm above liquid nitrogen and then lowered into the liquid nitrogen and stored. All samples were thawed by immersion for 30 sec in a 37ºC water bath and immediately evaluated. Progressive motility was best after Method III (63%), which was not different from fresh semen (94%) but was better (p< 0.05) than after Method II (25%) or Method I (6%). Forward velocity was again best after Method III (2.9) comparable to fresh semen (4.6) and Method II (2.5), but better (p< 0.05) than Method I (1.7). Morphological abnormalities were lowest after Method III, but were not significantly different from Methods II or I. Most common abnormalities were detached heads and bent, hairpin and coiled tails. It was concluded that slow, step-wise equilibration in tris-fructose-citrate extender followed by extender with ethylene glycol before freezing seems to produce the best progressive motility and forward velocity in frozen-thawed canine spermatozoa. | A utilização de sêmen congelado tornou-se extremamente comum na prática da inseminação artificial em cães, estimulando a pesquisa de métodos de congelação e diluidores. Neste estudo foram avaliadas as propriedades criopreservativas do etileno glicol na concentração final de 5%, utilizado em 3 diferentes protocolos de congelação. No Método I, uma parte de sêmen foi adicionada a duas partes de tris-frutose-ácido cítrico, contendo 7,5% de etileno glicol e congelado sem prévia refrigeração. No Método II, uma parte de sêmen foi adicionada a duas partes de tris-frutose-ácido cítrico contendo 7,5% de etileno glicol e resfrigerado até 5ºC por 1 hora antes da congelação. No Método III, uma parte de sêmen foi adicionada a uma parte de tris-frutose-ácido cítrico sem etileno glicol e refrigerado até 5ºC por 1 hora, sendo em seguida adicionada uma parte de tris-frutose-ácido cítrico, previamente refrigerado até 5ºC, contendo 15% de etileno glicol, mantido a 5ºC por mais 1 hora e congelado. Para a congelação em cada método, o sêmen foi envasado em palhetas plásticas de 0,5 ml, colocadas em vapor de nitrogênio a 5 cm da coluna líquida por 20 minutos, mergulhadas no nitrogênio líquido e armazenadas. Todas as amostras foram descongeladas em banho-maria a 37ºC durante 30 segundos e imediatamente avaliadas. O Método III não apresentou decréscimo significante da motilidade retilínea progressiva após a descongelação e apresentou os melhores resultados para o vigor espermático, comparando-se aos demais métodos. Entretanto, os 3 métodos não apresentaram diferença significativa com relação aos defeitos espermáticos pós-descongelação. Os tipos de defeitos mais freqüentemente encontrados foram cabeça solta normal, cauda dobrada, fortemente dobrada e fortemente enrolada. Foi possível concluir que o tempo de equilíbrio maior no diluidor tris-frutose-ácido cítrico seguido pela diluição em etileno glicol antes da congelação parece produzir para os espermatozóides de cães a melhor motilidade retilínea progressiva e vigor após a descongelação.

The objetive of this study was to identify the better dose between 300 (n = 20), 400 (n = 21) and 500 IU (n = 21) of FSH/LH to stimulate Nelore heifers. The superovulation treatment started on day 10 (D0 = estrous) of the estrous cycle in 8 decreasing aplications for 4 days. The embryo recovery was achieved on day 6.5 after the first artificial insemination. The superovulatory response for 300, 400 and 500 IU FSH/LH was follicles (15.12, 15.76 and 14.94); corpus luteum (10.68, 11.55 and 10.81) and transferable embryos (5.20, 1.81 and 2.76). The 300 IU of FSH/LH group presented the best results in regard to transferable embryos. The transferable embryos were cryopreserved by one-step method with 1.5 M of ethylene-glycol, resulting in 8 pregnancies (7.5%) of 106 embryos transferred by non-curgical method. The 300 IU of FSH/LH presented better superovulatory response in comparison with 400 and 500 IU in Nelore heifers. The transfer of Bos taurus indicus embryos cryopreserved by one-step method in 1.5 M of ethilene glycol was not efficient. | Este trabalho teve como objetivo identificar a dose mais eficiente de FSH/LH (300, 400 e 500 UI) no tratamento superovulatório de novilhas da raça Nelore, assim como avaliar o método one-step no processo de congelação de embriões. A variação da resposta superovulatória tem sido muito grande, o que explica o interesse de diversos pesquisadores em encontrar novos hormônios, doses e momentos para realizar a estimulação ovariana. Foram empregadas doses de 300 (n = 20), 400 (n = 21) ou 500 UI (n = 21) de FSH/LH, iniciando-se no décimo dia do ciclo estral, em 8 aplicações decrescentes, durante quatro dias consecutivos. Foi aplicado PGF2alfa concomitante com a quinta subdose de FSH/LH e realizadas duas inseminações artificiais às 12 e às 24 horas após o início dos sintomas de estro. As colheitas dos embriões foram realizadas 6,5 dias após a primeira inseminação artificial. Pelo exame ultra-sonográfico, avaliaram-se os números de folículos no momento da inseminação artificial (15,12; 15,76; e 14,94) e de corpos lúteos (10,68; 11,55; e 10,81) no dia da colheita, encontrando 5,20; 1,81; e 2,76 embriões viáveis, respectivamente, para 300 UI, 400 UI e 500 UI de FSH/LH. O grupo de 300 UI de FSH/LH apresentou os melhores resultados em relação aos embriões viáveis. Dos 106 embriões congelados pelo método one-step em 1,5 M de etilenoglicol e transferidos pelo método não-cirúrgico, 8 resultaram em prenhez (7,5%). A dose de 300 UI de FSH/LH apresentou melhor resposta superovulatória em comparação com as de 400 e 500 UI para novilhas da raça Nelore. A transferência de embriões Bos taurus indicus congelados pelo método one-step em 1,5 M de etilenoglicol não foi eficiente.

The effects of plunging temperature in liquid nitrogen and cryoprotectant dilution methods were evaluated for compacted mouse morulae frozen in 1.5 M ethylene-glycol (E), 1.5M propylene-glycol (P) or 1.4M glycerol (G). Morulae were equilibrated for 10 minutes in cryoprotectant solution and loaded into 0.25 ml straws with cryoprotectant solution in 3 columns (groups E1, P1, G1) or cryoprotectant in the center and PBS in the lateral columns (E2, P2). Straws were cooled at 0.5ºC/min to -25 or -30ºC and plunged into liquid nitrogen. Straws were thawed in water at 22ºC for 20 seconds. Cryoprotectant was diluted in 3 steps for group G1 and in one step for groups E1 and P1 (direct transfer to PBS + 10% FCS) and E2 and P2 (shaken to mix the 3 columns before transferring to PBS+ 10% FCS). Plunging temperature had no significant effect on the proportion of morulae developed to blastocysts in vitro; this proportion was higher (p < 0.0001) in E1 (69.2%) than in E2 (60.3%), G1 (51.9%) and combined for P1 and P2 (46.9%). In second experiment, the proportion of transferred morulae that developed to viable fetuses was lower (p < 0.07) for E1-25 than for E1-30, G1-30, E2-25 or unfrozen (control) embryos (8.7, 20.0, 20.0, 17.4 and 19.8%, respectively). In conclusion, the ethylene glycol diluted directly in PBS (E1) exhibit the highest rate of in vitro embryos development, but based on in vivo embryos development was more efficacious in plunging temperature at -30ºC (E1-30). | Os efeitos das temperaturas de imersão em nitrogênio líquido e dos métodos de remoção dos crioprotetores foram avaliados em mórulas de camundongos congeladas em etilenoglicol (E), propilenoglicol (P) e glicerol (G). Os embriões foram equilibrados em E (1,5M), P (1,5M) ou G (1,4M) por 10 minutos e envasados em palhetas de 0,25 ml com crioprotetor nas três colunas (E1, P1 G1) ou PBS nas colunas das extremidades (E2, P2). As palhetas foram resfriadas a 0,5ºC/minuto até -25 ou -30ºC e imersas em nitrogênio líquido. A descongelação dos embriões foi feita em água a 22ºC por 20 segundos. O crioprotetor dos embriões congelados em glicerol (Gl) foi removido em 3 etapas, dos congelados em etilenoglicol e propilenoglicol com crioprotetor nas 3 colunas (El e Pl) removido diretamente em PBS e dos congelados em etilenoglicol e propilenoglicol com PBS nas colunas das extremidades (E2, P2), após mistura das três colunas dentro da palheta, em PBS. Não houve influência da temperatura de imersão sobre o desenvolvimento embrionário in vitro, observando maior taxa (p < 0,0001) para El (69,2%) que para E2 (60,3%), Gl (51,9%) e a combinação P1 e P2 (46,9%). Para o desenvolvimento in vivo, a taxa de fetos foi menor (p < 0,07) para o grupo E1-25 do que para o Controle; Gl-30ºC; El-30ºC e E2-25ºC. Pode-se concluir que in vitro o melhor crioprotetor foi o etilenoglicol com remoção direta em PBS e que in vivo o etilenoglicol e o glicerol foram semelhantes a -30ºC.

Investigation of the efficiency of ethylene glycol and sucrose application in the capacity of cryoprotectors for cryopreservation of cattle embryos and their thawing in the conditions of application of saline solutions (as their dissolution medium) and nutritive media with a various structure was realized in the conditions of the Republic of Belarus. Research results showed that application of ethylene glycol in concentration 1,5 M and sucrose in concentration 1,0M proved to be the most effective. Regardless of the applied media the average safety of embryos was 93,8-96%, and acceptability - 59,3-62,5%. Peculiar feature of ethylene glycol use as cryoprotector for preservation of cattle embryos was that it could be quickly absorbed by a cell and quickly deduced from it. It made it possible to realize embryo transfer immediately after thawing. Application of ethylene glycol and sucrose as cryoprotectors could considerably simplify the procedure of transplantation of the frozen-thawed embryos, practically reducing it to a procedure of artificial insemination