Article

Kinetic and Temporal Factors Influence Chilling Injury to Germinal Vesicle and Mature Bovine Oocytes

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Abstract

In this study we examined the effects of low, above freezing temperatures on the viability and functionality of bovine oocytes. Germinal vesicle (GV) stage and in vitro matured oocytes (MII) were exposed to various combinations of time (15 and 60 min) and temperature (4, 16, 23, and 39 degrees C). After being treated, the ability of oocytes to undergo maturation and fertilization in vitro was examined, as well as their viability assayed by two fluorescent probes, fluorescein diacetate (FDA) and 5-carboxyfluorescein diacetate (cFDA). Cooling GV oocytes to 16 degrees C for 15 min reduced the fertilization rate by more than 40%, compared with those left at 39 degrees C. Surprisingly, cooling oocytes to 4 degrees C reduced the fertilization rate by only 10% compared with control. Exposing GV oocytes to temperatures below 23 degrees C reduced their viability. Similar to the reduction in fertilization, the viability of GV oocytes after exposure to 16 degrees C was reduced by more than 50%, whereas exposure to 4 degrees C reduced it by only 9%. Viability measurements using FDA and cFDA gave comparable results and showed a similar trend. The viability of MII oocytes and of GV oocytes pretreated with butylated hydroxytoluene, following exposure to low temperatures, was higher compared with that of GV controls. We interpret these results as indicating chilling effects on membrane integrity. Improving the chilling resistance of bovine oocytes may facilitate their short- and long-term preservation.

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... Estudos têm sido realizados visando facilitar o uso do gameta feminino (Arav et al., 1996) e, atualmente, a técnica mais promissora é a criopreservação de ovócitos, desejável por razões biológicas e comerciais (Shaw et al., 2000), considerando-se que o princípio do resfriamento consiste em reduzir a taxa metabólica e permitir a estocagem e transporte do ovócito (Zeron et al., 1999). A disponibilidade de ovócitos viáveis após a criopreservação permitiria maior flexibilidade em experimentos que exigem o uso simultâneo de grande número de gametas (Songsasen et al., 2002), aumentando a viabilidade de materiais para pesquisa básica (Otoi et al., 1997) e para os procedimentos da FIV (Saunders & Parks, 1999), além de permitir o estabelecimento de um banco de ovócitos destinado a preservar os recursos genéticos de espécies em risco de extinção (Lim et al., 1999;Ledda et al. 2001). ...
... Em geral, os ovócitos sofrem danos durante o processo de congelação, em decorrência de seu grande diâmetro e da elevada relação volume/superfície (Asada & Fukui, 2000), características que, associadas à penetração desigual do crioprotetor, podem resultar em danos em parte do ovócito e crioproteção insuficiente em outra. Também os ovócitos não têm sido efetivamente criopreservados, em virtude da sensibilidade da membrana citoplasmática, dos microtúbulos, do citoesqueleto, da zona pelúcida, dos cromossomos, das mitocôndrias e dos grânulos corticais a baixas temperaturas (Park et al., 1997;Zeron et al., 1999;Ledda et al., 2001) sendo que esses efeitos prejudiciais verificados em ovócitos de suínos, de humanos e bovinos (Hyttel et al., 1999;Wu et al., 1999;Isachenko et al., 2001). ...
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Objetivou-se avaliar os efeitos da criopreservação pelo método convencional em ovócitos imaturos e maturados in vitro. Utilizou-se ovócitos provenientes de ovários de vacas abatidas em matadouro, distribuídos em seis tratamentos: ovócitos não-congelados originados de células do cumulus oophorus (T1) e desnudados (T2) submetidos à MIV, FIV e CIV; ovócitos imaturos, originados de células do cumulus oophorus (T3) e desnudados (T4), congelados, reidratados, e ovócitos considerados normais, submetidos à MIV, FIV e CIV; ovócitos MIV, providos de células do cumulus oophorus (T5) e desnudados (T6), congelados, reidratados e submetidos à FIV e CIV. A congelação dos ovócitos foi realizada em soluções contendo 0,6; 1,2 e 1,8 mol L-1 de Etileno Glicol (EG) durante cinco minutos cada etapa. A descongelação foi realizada em banho-maria a 30 ºC por 20 segundos e, posteriormente, foram reidratados em três etapas (0,9 mol L-1 de EG + 0,3 mol L-1 de sacarose; 0,3 mol L-1 de sacarose, e sem EG e sem sacarose) de seis minutos. A principal alteração ultra-estrutural verificada nos ovócitos maturados in vitro e congelados foi a liberação prematura dos grânulos corticais e, tanto nos maturados quanto nos imaturos congelados, verificou-se vacuolização e redução das cristas mitocondriais. A taxa de maturação foi de 82,5; 75,4; 9,2 e 5,8%, para ovócitos do T1, T2, T3 e T4, respectivamente. As taxas de fecundação foram de 56,2; 0,0; 38,7; 8,6; 63,6 e 16,7% e de clivagem de 36,3; 7,9; 0,4; 0,0; 0,0 e 0,0%, para ovócitos do T1, T2, T3, T4, T5 e T6, respectivamente. Somente os ovócitos do T1 apresentaram desenvolvimento para mórulas e blastocistos (34,5%). Estes resultados indicam que as técnicas de congelação adotadas comprometeram a viabilidade do ovócito.
... In order to be effective the cooling technique should allow the transportation of embryos between distant cattle breeders and, therefore, a suitable medium is essential to extend the lifespan of embryos. Although phosphate buffered saline (PBS) supplemented with different concentrations of fetal calf serum (FCS) was the most commonly used cooling medium for bovine embryo handling (BonDurant et al., 1982;Lindner and Ellis, 1985;Pinto et al., 1999;Yang et al., 1991), the pregnancy rates even for in vivo-produced embryos were less than 50% (Lindner and Ellis, 1985). Therefore, some studies have been conducted in an attempt to develop an complex and efficient bovine embryo cooling medium that could extend the storage period without affecting its viability at the moment of ET (Ideta et al., 2013;Mori et al., 2006). ...
... However, as observed in our study and related by Otoi et al. (1999) the hatch rate was already lower in 48 h cooled blastocysts than in NC. It is well-known that IVP bovine embryos have less tolerance to the cryopreservation techniques than their in vivo counterparts (Abe et al., 2002;Villamil et al., 2012) which is particularly related to an excessive intracellular lipid droplets accumulation that can modify the physical and functional properties of membranes (Crocco et al., 2013) and result in significant cell damage when exposed to low temperatures, especially closed to 0°C, by direct chilling injuries (Arav et al., 1996;Zeron et al., 1999). Like proposed for cryopreservation of IVP embryos (Seidel Jr, 2006) we highlight the importance of modify the in vitro embryo production systems to render them more tolerant to storage under hypothermic temperatures, since it has proven possible to prolong the storage time under cooling of in vivo produced bovine embryos for up to seven and days (Ideta et al., 2013(Ideta et al., , 2015. ...
... They differ in their respective ability to control the process and in being field friendly or laboratory bound. These include, in increasing level of control over the cryopreservation process, pellet freezing (Gibson & Graham, 1969), dry-shipper (Roth et al., 1999), cold ethanol (Saroff & Mixner, 1955), liquid nitrogen vapour (Sherman, 1963;Roussel et al., 1964), controlled-rate automatic freezers (Landa & Almquist, 1979) and directional freezing (Arav, 1999). The alternative is a fast-cryopreservation technique called vitrification, also known as ice-free cryopreservation, a process in which liquid is transformed into an amorphous, glass-like solid, free of any crystalline structures (Luyet, 1937). ...
... The size of the oocyte is in the range of three to four orders of magnitude larger than the spermatozoa. The low surface-to-volume ratio and high intracytoplasmic lipid content make them very sensitive to chilling and highly susceptible to intracellular ice formation (Ruffing et al., 1993;Zeron et al., 1999;S. U. Chen & Yang, 2009). ...
Article
To preserve the world's biodiversity, the establishment of genome resource banks is desirable. These institutions can preserve gametes, embryos and tissues that, when needed, can be used to replenish a genetically dwindling population or species that are extinct in the wild or, theoretically, recreate an extinct species. A wide variety of options for genetic preservation has been developed over the short history of tissue and cell cryopreservation and success varies between classes and species. New technologies are still emerging; thus, when collecting samples, these should be of a wide variety to cover current and future technologies. Such samples can include (but should not be limited to) gametes, embryos, testicular and ovarian tissues, somatic cells or cell lines, and whole organs or whole animals. We will review the various current and emerging preservation technologies, and discuss their advantages and limitations.
... Estudos têm sido realizados visando facilitar o uso do gameta feminino (Arav et al., 1996) e, atualmente, a técnica mais promissora é a criopreservação de ovócitos, desejável por razões biológicas e comerciais (Shaw et al., 2000), considerando-se que o princípio do resfriamento consiste em reduzir a taxa metabólica e permitir a estocagem e transporte do ovócito (Zeron et al., 1999). A disponibilidade de ovócitos viáveis após a criopreservação permitiria maior flexibilidade em experimentos que exigem o uso simultâneo de grande número de gametas (Songsasen et al., 2002), aumentando a viabilidade de materiais para pesquisa básica (Otoi et al., 1997) e para os procedimentos da FIV (Saunders & Parks, 1999), além de permitir o estabelecimento de um banco de ovócitos destinado a preservar os recursos genéticos de espécies em risco de extinção (Lim et al., 1999;Ledda et al. 2001). ...
... Em geral, os ovócitos sofrem danos durante o processo de congelação, em decorrência de seu grande diâmetro e da elevada relação volume/superfície (Asada & Fukui, 2000), características que, associadas à penetração desigual do crioprotetor, podem resultar em danos em parte do ovócito e crioproteção insuficiente em outra. Também os ovócitos não têm sido efetivamente criopreservados, em virtude da sensibilidade da membrana citoplasmática, dos microtúbulos, do citoesqueleto, da zona pelúcida, dos cromossomos, das mitocôndrias e dos grânulos corticais a baixas temperaturas (Park et al., 1997;Zeron et al., 1999;Ledda et al., 2001) sendo que esses efeitos prejudiciais verificados em ovócitos de suínos, de humanos e bovinos (Hyttel et al., 1999;Wu et al., 1999;Isachenko et al., 2001). ...
Article
Full-text available
The objective of this study was to evaluate the cryopreservation of in vitro mature or immature oocytes by conventional method. The experiment was conducted using oocytes of cows' ovaries from slaughterhouse, distributed into six treatments: unfrozen oocytes with the cumulus oophorus cells (T1) and naked (T2) which were submitted to the process of MIV, FIV and CIV; immature oocytes, with the cumulus oophorus cells (T3) and naked (T4), which were submitted to the cryopreservation, unfrozen and the MIV, FIV and CIV; oocytes with the cumulus oophorus cells (T5) and naked (T6), which were submitted to in vitro maturation, cryopreservation, and FIV and CIV. The oocytes were frozen by the conventional methods, dehydrated by the immersion in three solutions with 0.6; 1.2 and 1.8 mol L-1 of Ethylene Glycol (EG) during 5 minutes each phase. The thawing phase was done by the immersion in water-bath at 30 ºC during 20 seconds, and so, the oocytes were re-hydrated in three phases (0.9 mol L-1 EG + 0.3 mol L-1 of sacarose; 0.3 L-1 of sacarose without EG and sacarose) for six minutes each one. The mainly ultrastructural changes in cryopreserved matured oocytes were prematurely released of cortical granules. However, the frozen immature and mature oocytes showed vacuolization and disappearance of cristae mitochondrial. The frozen immature oocytes showed the maturation rate of 82.5, 75.4, 9.2 and 5.8% for T1, T2, T3 and T4, respectively. The fecundation rate were 56.2, 0.0, 38.7, 8.6, 63.6 and 16.7% and from the cleavage were 36.3, 7.9, 0.4, 0.0, 0.0 and 0.0% for T1, T2, T3, T4, T5 and T6, respectively. Morula and blastocyst were observed only for unfrozen and naked oocytes (T1) (34.5%). These results showed that the frozen protocols affect the viability of the oocytes.
... The integrity of the bovine oocyte membrane is generally altered during cooling (Arav et al., 1996;Horvath and Seidel, 2006;Seidel, 2006;Spricigo et al., 2012), and may contribute to the lowered fertilization rate of cryopreserved oocytes (Arav et al., 1996;Zeron et al., 1999). This cryopreservation-dependent oocyte membrane damage could leave membrane-bound CD9 susceptible to damage. ...
... For assessment of viability, oocytes in Control, Toxicity, and Vitrification groups were first washed three times in maturation medium, incubated for 30 min in a CO 2 incubator, and then quickly washed three times in DPBS solution. The oocytes were then cultured in DPBS containing 2.5 mg/ml fluorescein diacetate for 3 min at 38.58C, and then washed three times in DPBS solution before examining the membrane integrity under an inverted fluorescence microscope according to the method of Zeron et al. (1999). High intensity fluorescence was considered high viability, low intensity was regarded as low viability, and nonfluorescence indicated the oocytes were not viable. ...
Article
This study was conducted to investigate the effect of vitrification of bovine metaphase-II (MII) oocytes on CD9 expression and fertilization capacity. Surviving vitrified/warmed oocytes were used to detect CD9 distribution (fluorescence microscopy), CD9 mRNA (qRT-PCR), and CD9 protein expression (Western blot), and to analyze in vitro fertilization rates (number of sperm bound to, or penetrated, the oocytes) after removing the zona pellucida. Fresh oocytes acted as control. The experimental results showed that the vitrification/warming procedures significantly decreased CD9 expression at the mRNA and protein levels, and changed the CD9 distribution pattern in bovine oocytes. After fertilization in vitro, the average number of sperm binding and penetration of vitrified oocytes were significantly lower than those of the non-vitrified oocytes. In conclusion, vitrification of bovine oocytes caused a decrease in CD9 expression at the mRNA and protein levels, and an alteration of CD9 distribution pattern, which may have resulted in lowered fertilization capacity. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.
... The phenomenon of recrystallization of both intracellular and extracellular ice, in frozen samples, occurs as smaller ice crystals with a rate of recrystallization that increases with increasing temperature [13]. It has largely been reported that chilling injury can modify the structure and integrity of plasma membranes [19, 20] mainly composed by phospholipids and cholesterol [21]. Even though high concentrations of cholesterol and polyunsaturated fatty acids give more fluidity to the membrane at lower temperature [22], during cryopreservation the cooling process causes phase transition of membrane lipids and impairs membrane protein function. ...
Article
Full-text available
Cryopreservation of human spermatozoa-introduced in the 1960's-has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.
... Common cryopreservation strategies include: (a) slow freezing (Mandawala, Harvey, Roy, & Fowler, 2016;; (b) vitrification (Zeron, Pearl, Borochov, & Arav, 1999); ...
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Extracellular vesicles (EVs) contain specific proteins, lipids, and nucleic acids that can be passed to other cells as signal molecules to alter their function. However, there are many problems and challenges in the conversion and clinical application of EVs. Storage and protection of EVs is one of the issues that need further research. To adapt to potential clinical applications, this type of problem must be solved. This review summarizes the storage practices of EVs in recent years, and explains the impact of temperature on the quality and stability of EVs during storage based on current research, and explains the potential mechanisms involved in this effect as much as possible.
... However, unique aspects of the oocyte make it a notoriously difficult cell to cryopreserve. For example, the volume-tosurface ratio of the oocyte is greater than other cells, thus complicating the dehydration process [5,73,74,100]. Also, membrane characteristics and permeability differences appear to make the oocyte more sensitive to chilling injury [73,97]. ...
Article
Full-text available
Human oocyte cryopreservation - An emerging ART technique: Are we heading in the right direction? Murid Javed, DVM, PhD1, Navid Esfandiari, DVM, PhD2, Jason Swain, PhD3 and Essam Michael, MD, FRCS (C)4 1,4 Astra Fertility Centre, 4303 Village Center Court, Mississauga, ON, L4Z 1S2, Canada murid.javed@astrafertility.com 2Toronto Centre for Advanced Reproductive Technology, 150 Bloor St. (W), Toronto, M5S 2X9 and Department of Ob/Gyn, University of Toronto, ON, Canada. Navid.esfandiari@utoronto.ca 3Dept. Ob/Gyn, Center for Reproductive Medicine, University of Michigan, 475 Market Place Ste B, Ann Arbor, MI 48108, USA. swainj@umich.edu Abstract Oocyte cryopreservation is a promising adjunct to human assisted reproductive technology. Slow rate freezing has been the cryopreservation standard for storage of sperm, embryos and, subsequently, oocytes. However, earlier concerns regarding damage to the meiotic spindle, loss of cortical granules and the low success rates as compared to the relative success of embryo cryopreservation caused little interest until the 1990s. Interest increased when many studies indicated that acceptable oocyte survival, in vitro fertilization, normal embryos and adequate blastocyst development can be achieved with oocyte cryopreservation. Recently introduced oocyte vitrification techniques are proving to be more efficient. Survival rates are close to 100% and developmental rates are similar to those achieved with fresh oocytes. This efficiency opens the way to the widespread application of the technique in various medical, legal, and social situations, even to replace embryo cryopreservation with the oocyte cryopreservation. Oocyte vitrification has dominated slow freezing to such an extent that it may soon become the exclusive cryopreservation choice, especially considering that potential disease transmission problems commonly associated with vitrification due to direct exposure of oocytes to liquid nitrogen can be eliminated by using the proper techniques and devices. Furthermore, cryopreservation of immature oocytes, ovarian follicles, ovarian tissue and whole ovary are other emerging technologies. Oocyte cryopreservation has tremendous opportunity for preserving fertility in cancer patients, for those who may not have sperm following oocyte retrieval and for those women who wish to delay their motherhood. The purpose of this article is to review the history of oocyte cryopreservation, its applications, current cryopreservation techniques and future trends for fertility cryopreservation, to determine if oocyte cryopreservation is proceeding in the right direction.
... On thawing, ice crystals can cause mechanical damage to the subcellular organelles. Various studies have shown that the damage induced by cryopreservation can affect the integrity of the plasma membrane (Arav et al. 1996;Zeron et al. 1999), which contains phospholipids and cholesterol (Giraud et al. 2000). The latter authors showed that the freeze-thaw process induces a rigidifying effect on the sperm membrane and suggested that the adaptability of mammalian sperm to freeze-thaw-induced stress, and hence its ability to withstand cryopreservation, might depend on the fluidity of its membrane, which is in turn modulated by the membrane's lipid composition (Giraud et al. 2000). ...
Article
Cryopreservation is a technique that can keep sperm alive indefinitely, enabling the conservation of male fertility. It involves the cooling of semen samples and their storage at −196°C in liquid nitrogen. At this temperature all metabolic processes are arrested. Sperm cryopreservation is of fundamental importance for patients undergoing medical or surgical treatments that could induce sterility, such as cancer patients about to undergo genotoxic chemotherapy or radiotherapy, as it offers these patients not only the hope of future fertility but also psychological support in dealing with the various stages of the treatment protocols. Despite its importance for assisted reproduction technology (ART) and its success in terms of babies born, this procedure can cause cell damage and impaired sperm function. Various studies have evaluated the impact of cryopreservation on chromatin structure, albeit with contradictory results. Some, but not all, authors found significant sperm DNA damage after cryopreservation. However, studies attempting to explain the mechanisms involved in the aetiology of cryopreservation-induced DNA damage are still limited. Some reported an increase in sperm with activated caspases after cryopreservation, while others found an increase in the percentage of oxidative DNA damage. There is still little – and contradictory – information on the mechanism of the generation of DNA fragmentation after cryopreservation. More studies are needed to establish the true importance of such damage, especially to improve the results of ART.
... Despite the heightened interest in oocyte preservation, many challenges in its application remain. For example, the volume of oocytes is much larger than that of spermatozoa, thus substantially decreasing the surface-to-volume ratio and making oocytes more sensitive to chilling and highly susceptible to intracellular ice formation (Toner et al. 1990;Ruffing et al. 1993;Arav et al. 1996;Zeron et al. 1999). Moreover, the removal of cumulus cells, which accelerate capacitation of spermatozoa, and the freezing procedure itself often cause significant reduction of the fertilisation rate (Carroll et al. 1990;Vincent et al. 1990). ...
Article
The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation-activation, embryonic development of oocytes-zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze-thawing did not affect the later development of zygotes.
... However, unique aspects of the oocyte make it a notoriously difficult cell to cryopreserve. For example, the volume-tosurface ratio of the oocyte is greater than other cells, thus complicating the dehydration process [5,73,74,100]. Also, membrane characteristics and permeability differences appear to make the oocyte more sensitive to chilling injury [73,97]. ...
Article
Full-text available
Purpose: To highlight emerging techniques aimed at improving oocyte cryopreservation. Methods: Review of available and relevant literature through Pubmed and Medline searches. Results: Oocyte cryopreservation is an increasingly common procedure utilized for assisted reproduction and may benefit several patient populations. Therefore, improving efficiency is paramount in realizing the tremendous promise of this approach. However, in addition to numerous studies looking to improve oocyte cryopreservation efficacy via examination of variables involved with protocol methodology, such as type/concentration of cryoprotectant (CPA), type of storage device, or cooling/warming rates, there are more novel approaches for improvement. These alternate approaches include utilizing different the stages of oocytes, examining alteration of basal media and buffer composition, optimizing CPA exchange protocols and device loading through use of automated technology, as well as examination/manipulation of oocyte cellular composition to improve cryotolerance. Finally, elucidating more accurate or insightful indicators of "success" is crucial for continued improvement of oocyte cryopreservation. Conclusion: Oocyte cryopreservation has improved dramatically in recent years and is receiving widespread clinical use. Novel approaches to further improve success, as well as improved methods to assess this success will aid in continued improvement.
... This method is effective for selection of live porcine oocytes without affecting their in vitro developmental competence (Shi et al. 2006). In brief, oocytes were cultured in Hepes-PXM containing 2.5 mg/mL FDA for 2 min at 38.5°C and then washed three times in Hepes-PXM before examination of membrane integrity under an inverted fluorescence microscope according to the method by Zeron et al. (1999). FDA was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (2.5 mg/mL) and kept at -20°C until just before use. ...
Article
Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm-penetrated oocytes were observed to some extent (30-40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two-cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP3 R1), the channel responsible for Ca(2+) release during IVF in porcine oocytes. Localization of IP3 R1 close to the plasma membrane and total expression level of IP3 R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified-warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP3 R1 by the vitrification procedure.
... Lipid phase transition (LPT) in cell membranes are also responsible for chilling injury in mammalian sperm [11] and oocytes [3]. At the temperature around phase transition, chilled membranes lose fluidity and become leaky, which causes damage to cells [44]. ...
Article
Methanol is a widely used cryoprotectant (CPA) in cryopreservation of fish embryos, however little is known about its effect at the molecular level. This study investigated the effect of methanol on sox gene and protein expression in zebrafish embryos (50% epiboly) when they were chilled for 3h and subsequently warmed and cultured to the hatching stages. Initial experiments were carried out to evaluate the chilling tolerance of 50% epiboly embryos which showed no significant differences in hatching rates for up to 6h chilling in methanol (0.2-, 0.5- and 1M). Subsequent experiments in embryos that had been chilled for 3h in 1M methanol and warmed and cultured up to the hatching stages found that sox2 and sox3 gene expression were increased significantly in hatched embryos that had been chilled compared to non-chilled controls. Sox19a gene expression also remained above control levels in the chilled embryos at all developmental stages tested. Whilst stable sox2 protein expression was observed between non-chilled controls and embryos chilled for 3h with or without MeOH, a surge in sox19a protein expression was observed in embryos chilled for 3h in the presence of 1M MeOH compared to non-chilled controls and then returned to control levels by the hatching stage. The protective effect of MeOH was increased with increasing concentrations. Effect of methanol at molecular level during chilling was reported here first time which could add new parameter in selection of cryoprotectant while designing cryopreservation protocol. Copyright © 2015. Published by Elsevier Inc.
... The cryotolerance of embryos derived from in vitro fertilization (IVF) is not constant, and depends in part on culture conditions [4,5]. During the process of cryopreservation, embryonic cells are exposed to mechanical, thermal and chemical stresses, and a chilling injury that occurs during the cooling process may alter the structure of cell membranes [6,7]. ...
Article
The ATP-binding cassette sub-family B member 1 (ABCB1) plays a critical role in maintaining the metabolic capability of cells as an efflux transporter that pumps xenobiotics out of cells. We investigated the effects of highly expressed ABCB1 on the development and viability of cryopreserved bovine embryos. TheABCB1 level in cultured bovine embryos was decreased during development to blastocyst-stage compared to germinal vesicle- and second metaphase-stage oocytes. When bovine embryos were cultured with forskolin and/or rifampicin, the ABCB1 level was significantly increased in blastocysts but embryo development was not significantly improved. After embryo cryopreservation, highly ABCB1-expressed blastocysts exhibited significant increases in viability and hatching rates. The high viability of the cryopreserved blastocysts was accompanied by a significant increase in cell proliferation during culture for 48h.Thus, ABCB1 is expressed in bovine oocytes and embryos, and the cellular quality of bovine blastocysts is improved by the enhancement of ABCB1 expression.
... It was widely reported that several damaging processes such as thermal shock with formation of intracellular and extracellular ice crystals, cellular dehydration, and osmotic shock could occur during freezing-thawing of human spermatozoa (Stanic et al., 2000). It has also been reported that chilling injury can modify the structure and integrity of plasma membranes (Arav et al., 1996;Zeron et al., 1999) mainly composed by phospholipids and cholesterol (Giraud et al., 2000). Even though high concentrations of cholesterol and polyunsaturated fatty acids give more fluidity to the membrane at lower temperature (Quinn, 1985), during cryopreservation the cooling process causes phase transition of membrane lipids and impairs membrane protein function. ...
... However, other studies report that the ability of oocytes to survive cryopreservation is not affected by the meiotic stage in which the oocyte was cryopreserved (Le Gal and Massip 1999). Differences among these studies can be partially explained by the use of different CPAs because of the selective tolerance of oocytes at different meiotic stages (Zeron et al. 1999). ...
Article
This review presents some of the most noticeable aspects related with the oocyte cryopreservation procedures, emphasizing their evolution in the bovine, which points towards the critical points determining the reduced survival rates of female gametes to freezing and vitrification. Factors such as the maturation status, the cytoskeleton and membrane sensitivity, the role of the cumulus cells, the impact of the cryoprotectants agents and the protocols utilized and the future of this tool have been extensively reviewed.
... Chilling injury has been linked to damages to meiotic spindles, microtubules, and microfilaments of mammalian oocyte (Wu et al., 1999;Zenze et al., 2001;Songsasen et al., 2002). At low temperatures microtubules are depolymerised, and cellular processes including cell division in oocytes can be irreversibly disrupted (Magistrini and Szollosi 1980;Martino et al., 1996); the plasma membrane can suffer lateral phase separation (Arav et al., 1996;Zeron et al., 1999;Arva et al., 2000); proteins can be denatured due to the destabilization of hydrophobic bounds (Dinnyes et al., 1998); the cell membrane shrinkage relative to the intracellular space, may result in stress and damage to the membrane (McGrath, 1987); and a disorder of metabolic and enzymatic processes due to their inhibition at sub-physiological temperature can occur (Mazur et al., 1992;Browne et al., 2001) which would be especially detrimental in embryonic development in some teleosts. ...
... The COC viability was evaluated by 5-carboxyfluorescein diacetate (cFDA, Sigma) and trypan blue staining [19,20]. After collection, the oocytes were incubated for 1 h, denuded and exposed (10 min, 32 8C) to synthetic oviductal fluid (SOFaaBSA) containing 11 mg/mL cFDA. ...
Article
The aim of the present study was to examine the effects of superoxide dismutase (SOD) addition to the ovary transport medium (4°C, 3-72h) on ovarian cell viability and apoptosis and in vitro embryo production (IVEP) in domestic cats. The ovaries collected from 76 mixed-breed domestic queens were randomly assigned to the control or SOD-treated groups and incubated for 3, 24, 48 or 72h. The ovaries were then subjected to the following: (1) fixed in formalin to assess the incidence of apoptosis (fragmented DNA in situ detection kit), (2) stored at -196°C in liquid nitrogen to evaluate the expression of the pro-apoptotic Bax gene and the anti-apoptotic Bcl-2 gene (RT-PCR), and (3) used to obtain the cumulus-oocyte complexes (COCs) in order to test the cell viability (carboxyfluorescein or trypan blue staining) and IVEP. The incidence of apoptosis appeared to be higher in the control compared with the SOD-treated ovaries. The ovarian expression of Bax was lower and the Bcl-2 expression was higher in the SOD-treated group compared with the control group. The presence of SOD in the transport medium increased the viability of COCs and IVEP compared with the control medium. In summary, the supplementation of the ovary transport medium with SOD reduced cellular apoptosis and enhanced COC survival and IVEP in domestic cats. Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
... Consecuencias de la vitrificación (crioinjurias): En contraste de lo que ocurre con los embriones bovinos producidos in vivo, los embriones producidos in vitro presentan un mayor daño y una capacidad disminuida de sobrevivencia después de la criopreservación (Gómez et al. 2009); sin embargo, mediante el uso de la vitrificación las lesiones producidas, se reducen considerablemente. Los embriones cuando son vitrificados pueden sufrir daños morfológicos y funcionales, denominados crioinjurias (Zeron et al. 1999; Vajta & Kuwayama, 2006). Dentro de las crioinjurias, se encuentran alteraciones en la permeabilidad de la membrana celular, por la formación de cristales de hielo (Leibo et al. 1996), fragmentación del ADN, daño en el huso meiótico, apoptosis celular (Park et al. 2006) y expresión génica alterada (Stinshoff et al. 2011). ...
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RESUMEN En Colombia y en el mundo la producción de embriones in vitro, a nivel comercial, se ha incrementado en los últimos años. La transferencia de los mismos, se realiza, principalmente, en fresco, debido a las posibles lesiones causadas por los procesos de criopreservación. La vitrificación es una herramienta útil en tecnologías de reproducción asistida (TRA), que se emplea en medicina humana y en veterinaria, que puede ser usada como alternativa para la criopreservación de germoplasma (oocitos, espermatozoides y embriones). Pese a los grandes avances que se han realizado en el desarrollo de dicha técnica, su aplicación es escasa en la industria de producción de embriones bovinos y se emplea, básicamente, en proyectos de investigación. En esta revisión, se discuten diferentes aspectos involucrados en el proceso de vitrificación de embriones in vitro, algunos de los resultados obtenidos, las ventajas y las principales limitantes de la misma. SUMMARY Around the world and in Colombia the in vitro cattle embryo production has increased in the last years. The embryo transfer of in vitro produced embryos is mainly realized with fresh embryos, due to the possible injuries induced during the cryopreservation process. Vitrification is a tool applied in assisted reproductive technologies (ARTs) and it is used as an alternative for germplasm cryopreservation (sperm, oocytes and embryos) in human and veterinary medicine. Despite major efforts focused on the development of the technique, its commercial application is scarce in the embryo production industry and it is employed mainly in research. In this review, different aspects involved in the in vitro embryo vitrification technique, such as protocols, main advantages, limitations and some results, are discussed.
... The purpose of cryopreservation procedures is to minimize the damage and help cells to regenerate. However, there are many problems associated with oocyte cryopreservation related to the injury or sensitivity due to chilling and to the toxicity of cryoprotectants that cause considerable morphological and functional damages (Succu et al., 2007;Zeron et al., 1999). In recent years, vitrification has been adopted by many researchers as an alternative promising method assuring minimum freezing injuries to the cells and higher survival rates of oocytes after thawing in compared to slow rate freezing method. ...
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The aim of the present investigation was to establish a reliable vitrification protocol for camel o ocytes. A total of 540 cumulus o ocyte complex es (COCs) were collected by manual slicing and divided into two groups. Three hundred o ocytes were used to investigate the effect of the open pulled straw (OPS) vitrification procedure on th e maturation rate of vitrified immature camel o ocyte s compared to control (non - vitrified). The rest ( 240 o ocytes ) were employed to evaluate the effect of vitrification on the morphological characteristics of immature and mature camel o ocytes . C OCs were treated with vitrification solution - 1 (VS - 1)  10% Dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG)  for 30 - 45 s ec and transferred to vitrification solution - 2 (VS2 )  20% DMSO, 2 0% EG and 0.5M sucrose  . Oocytes were loaded into t he OPS and then directly submerged into liquid nitrogen (LN 2 ) for 1h. T he average value of maturation rate between the vitrified immature camel o ocytes and the non - vitrified group ( control ) revealed non - s ignificant differences as indicated by expansion of o ocytes (88% (132/150 ), and 85.3% (128/150), respectively) . However, the average rate of extrusion of the first polar body was significantly reduced in the vitrified immature o ocytes (20.3%) compared to control group (40.1%). In addition, the morphological abnormalities occurred at higher rate in vitrified immature (GV stage) o ocytes t han in vitrified mature o ocytes  16/99 (13.6%) vs, 6/106 (6.4%), respectively  . C onsequently , the survival rate was significantly reduced in vitrified immature o o cytes compared to vitrified mature o ocytes (83.9% vs. , 92.4%, respectively). In conclusion , the present study revealed that the camel o ocytes could be successfully cryopreserved by OPS vitrification using EG and DMSO as cryoprotectant . Moreover, our results demonstrated that in camel mature o ocytes are mor e resistant to cryoinjuries than immature o ocytes and could produce a high percentage of normal o ocytes that could be useful for future use in in vitro fertilization and camel improve ment programs.
... On thawing, ice crystals can cause mechanical damage to the subcellular organelles. Various studies have shown that the damage induced by cryopreservation can affect the integrity of the plasma membrane (Arav et al. 1996;Zeron et al. 1999), which contains phospholipids and cholesterol (Giraud et al. 2000). The latter authors showed that the freeze-thaw process induces a rigidifying effect on the sperm membrane and suggested that the adaptability of mammalian sperm to freeze-thaw-induced stress, and hence its ability to withstand cryopreservation, might depend on the fluidity of its membrane, which is in turn modulated by the membrane's lipid composition (Giraud et al. 2000). ...
Chapter
Cryopreservation is a technique that can keep sperm alive indefinitely, enabling the conservation of male fertility. It involves the cooling of semen samples and their storage at −196 °C in liquid nitrogen. At this temperature all metabolic processes are arrested. Sperm cryopreservation is of fundamental importance for patients undergoing medical or surgical treatments that could induce sterility, such as cancer patients about to undergo genotoxic chemotherapy or radiotherapy, as it offers these patients not only the hope of future fertility but also psychological support in dealing with the various stages of the treatment protocols.
... The cryopreservation technique is basically categorized into three: programmable slow freezing, vitrification, and low CPA vitrification [116]. In the programmable slow freezing process, most cells are frozen at 1 °C, which minimizes ice injury to support cell survival [117], since oocytes are prone to cell injury [118]. In the vitrification process, a higher concentration of cryoprotective agents (CPAs) is used to prevent ice formation. ...
Article
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With the recent development in science and technology, the advancement in in vitro fertilization to treat infertility has been one of the most revolutionary advances. However, the success rate of the IVF process depends on the efficiency of each step of the process. The physical tools used to enhance the process continue to change. Microfluidics technology is an emerging technology being used in multiple biological applications for the miniaturization and specification of laboratory techniques. Technology is used along with IVF to enhance the outcome by facilitating every step of the process. Microfluidics can be used to handle gametes, culture embryos, cryopreservation, and for many other applications. This review will highlight the applications of microfluidics in different stages of IVF, including the handling of gametes, sperm collection and isolation, sperm sorting, embryo culture, cryopreservation and the fabrication process of microfluidics, focusing on the benefits and shortcomings of these applications.
... Entretanto, as estruturas produzidas atualmente são deficitárias e mais sensíveis à criopreservação do que os produzidos in vivo [6,9,10,16,19]. Isto torna o método convencional de congelamento inadequado para a criopreservação de embriões PIV [8,16,19], principalmente em função do excessivo tempo de exposição à faixa térmica correspondente à solidificação dos lipídios [31]. A metodologia mais promissora para criopreservar esses embriões é a vitrificação [2,7,16,22,24,25], que em função de sua maior velocidade de resfriamento e aquecimento proporciona uma passagem rápida pela faixa crítica de temperatura. ...
... Extracellular and intracellular ice crystal generation, osmotic shock and cellular dehydration have been reported after freezing and thawing of human sperms [9]. Cooling damage can also change the integrity and structure of cellular membranes [10,11,12]. Both the plasma membranes and the mitochondrial membranes have this sensitivity to cryopreservation [13]. ...
... The membrane integrity of vitrified-warmed embryos under a microscope according to the method by Zeron et al [29]. Embryos with normal and spherical shape, without lysis, and not shrunken, swollen, or blackened were regarded as surviving. ...
Article
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Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated ε-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p
... The practical benefits of vitrification to preserve bovine oocytes are nevertheless limited since vitrified oocytes show impaired in vitro maturation and early embryo development. PLOS During oocyte cryopreservation, cooling and osmotic stress can cause irreversible damage to membrane integrity [2,3]. Oocytes undergo substantial volume changes due to water and cryoprotectant movement during cryopreservation (reviewed in: [4]). ...
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This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage.
... Considerable morphological and functional damages can be induced by the low temperatures and the exposure to CPs during oocytes cryopreservation (Son et al., 1996;Zeron et al., 1999;Paynter, 2005). In human oocytes, changes have been observed in the glycoprotein structure of the ZP due to the premature release of cortical granules, which can be responsible for a decreased fertilisation rate (Mandelbaum, 1991;Ghetler et al., 2006). ...
... Results from a retrospective study of 11,768 cryopreserved human embryos that underwent at least one thaw cycle from 1986 to 2007 showed that A c c e p t e d M a n u s c r i p t Page 8 there was no significant impact of the duration of storage on clinical pregnancy, miscarriage, implantation, or live birth rate, whether from IVF or oocyte donation cycles. 41 Since oocytes are highly prone to chilling injury, 10 cryopreservation of immature oocytes and ovarian tissue is a promising approach, with reports of live births, but the need for investigational improvement remains. 37, 42-45 ...
Article
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Cryopreservation is a process that preserves organelles, cells, tissues, or any other biological constructs by cooling the samples to very low temperatures. The responses of living cells to ice formation are of theoretical interest and practical relevance. Stem cells and other viable tissues, which have great potential for use in basic research as well as for many medical applications, cannot be stored with simple cooling or freezing for a long time because ice crystal formation, osmotic shock, and membrane damage during freezing and thawing will cause cell death. The successful cryopreservation of cells and tissues has been gradually increasing in recent years, with the use of cryoprotective agents (CPAs) and temperature control equipment. Continuous understanding of the physical and chemical properties that occur in the freezing and thawing cycle will be necessary for the successful cryopreservation of cells or tissues and their clinical applications. In this review, we briefly address representative cryopreservation processes, such as slow freezing and vitrification, and the available CPAs. In addition, some adverse effects of cryopreservation are mentioned.
Book
The biblical story of Noah's Ark has a humanitarian and educational purpose. It is there to teach us that we should show respect to mother earth by protecting all species on the planet from disaster that may lead to their extinction. One of the key aspects of this endeavor is to strive to preserve animal fertility. Two disciplines work in tandem to facilitate this goal - biotechnology of reproduction and cryobiology. This book reviews various achievements in this field and discusses emerging technologies in cryopreservation of gametes, embryos, and gonads of humans and domestic, laboratory and wild animals.
Article
Bovine oocytes are less likely to undergo successful cryopreservation than cleavage-stage embryos. Bovine oocytes characteristically contain high levels of lipids that represent one of the major obstacles limiting efficient cryopreservation. These droplets together with structures such as cumulus cells, zona pellucida, cytoplasm membrane, cortical granules, mitochondria, spindle, and cytoskeleton (microtubles and microfilaments) often incur serious damage during cooling and warming. The cryoinjury could, to some extent, be decreased by selection of proper permeable and non-permeable cryoprotectants, and of vitrification with high cooling and warming rates. Additionally, such measures may also enhance their cryotolerance as partial removal of cumulus cells, modification of oocyte membrane constituents, polarization of the cytoplasmic lipid droplets by centrifugation, and addition of cytoskeleton relaxants or ice blockers into vitrification solutions. The improvement in cryopreservation methodology for bovine oocytes will no doubt augment other technologies such as bovine cloning and the establishment of gene bank for transgenic cattle.
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Cryopreservation technology regarding banded coral shrimp (Stenopus hispidus) embryos is important as it could improve cultivation and preservation of the species. The development of this technology is to reduce collections of this species from the wild, thus preventing damage to coral reefs. This study investigated the tolerance of different developmental stages of S. hispidus embryos in response to low temperature in the presence or absence of cryoprotectant. Embryos undergoing three stages of embryonic development (eye-formation, heart beat and pre-hatch stage) were selected and exposed to 5, 0 and -5°C in cryoprotectant solutions of 1 or 2M methanol for 1, 2, 4, 6, 8, 16 and 32h. Embryo survival was evaluated based on their hatching percentage. In experiments on the effect of different concentrations of methanol on chilling sensitivity of embryos, it was determined that methanol at 1M methanol reduced the chilling sensitivities of embryos most effectively when compared to the other tested concentrations. Experiments regarding the chilling sensitivity of embryos in different developmental stages indicated that pre-hatch stage embryos were more resistant to subzero temperatures than early stage embryos; they tolerated the 32h exposure at 5 and 0°C without a loss in survival. The study also indicated that late stage embryos are considered to be resistant to chilling, and that pre-late stage embryos are better candidate for cryopreservation.
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During the past 15 years, embryo transfer (ET) has become increasingly widespread within the sport-horse breeding industry. At present, however, the vast majority (>95%) of horse embryos are transferred fresh or after chilled storage for up to 24 h, whereas cryopreservation is rarely employed despite its obvious potential for simplifying recipient mare management and facilitating long-term storage and international transport of embryos. A number of inter-related factors have contributed to the slow development and implementation of equine embryo cryopreservation, and these include the following: (i) the absence of commercially available products for reliably stimulating superovulation; (ii) very poor pregnancy rates following cryopreservation of embryos >300 μm in diameter; (iii) difficulty in recovering embryos at early developmental stages amenable to cryopreservation; and (iv) interembryo variation in susceptibility to cryodamage. However, acceptable success rates (>55% pregnancy) have been reported for both slow-frozen and vitrified small embryos (<300 μm), and there is renewed interest in cryopreservation, not only in the context of standard ET programmes, but also because it would facilitate pre-implantation genetic testing and allow wider access to techniques for producing embryos in vitro, such as intracytoplasmic sperm injection and nuclear transfer. This article will review the current status of equine embryo cryopreservation.
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A bed sore, pressure sore, pressure ulcer and decubitus ulcer are synonymous. As the name signifies this is the result of constant pressure on skin. This constant pressure occurs in bed ridden patients and paraplegics continuously sitting in chair. All patients who are immobile for long time due to spinal injury, paralysis, and major surgery or unconscious are likely to get bed sores. Geriatric patients are having dry, non-elastic thin skin with poor protein energy malnutrition and multiple vitamin deficiency are more prone to bed sores formation. These old age patients are less to change position due to dementia or sedatives and thus increasing the risk of bed sore. Patients with poor bladder and bowel control having wet clothes and beddings can also suffer from bedsores. The treatment strategies in homecare of bedsore are: education of family members, assessment of skin likely to develop bedsore, staging of bedsore, treatment strategies of bedsore, nutritional management, psychological management, care of urine and stool management. Keywords: Bedsore, pressure sore, homecare, domiciliary care, nursing care, self-care of bedsore
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Nanotechnology is the study of extremely small structures, having size of 0.1 to 100 nm. Nano medicine is a relatively new field of science and technology. Brief explanation of various types of pharmaceutical nano systems is given. Classification of nano materials based on their dimensions is given. An application of Nanotechnology in various fields such as health and medicine, electronics, energy and environment, is discussed in detail. Applications of nano particles in drug delivery, protein and peptide delivery, cancer are explained. Applications of various nano systems in cancer therapy such as carbon nano tube, dendrimers, nano crystal, nano wire, nano shells etc. are given. The advancement in nano technology helps in the treatment of neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease. Applications of nano technology in tuberculosis treatment, the clinical application of nanotechnology in operative dentistry, in ophthalmology, in surgery, visualization, tissue engineering, antibiotic resistance, immune response are discussed in this article. Nano pharmaceuticals can be used to detect diseases at much earlier stages. Keywords: Nano devices, nano material, nano medicine, nano pharmaceutics, drug delivery
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This study was conducted in an effort to improve our understanding of the response of Asian elephant (Elephas maximus, Em) spermatozoa to chilling. Semen was collected from two elephant bulls by means of the manual rectal stimulation method. Five out of seven semen collections were deemed to be suitable for use based on motility (ranging from 20% to 60%) and membrane integrity. We evaluated the chilling sensitivity by incubating the sperm with a fluorescent dye (5-carboxyfluorescein diacetate (cFDA)) at 16°C, 12°C, 4°C, and 22°C (control). Cells with an intact membrane retained the dye and were identified as viable. The membrane lipid phase transition (LPT) temperature curve was determined with a Fourier transform infrared (FTIR) spectrometer connected to an FTIR microscope. The LPT center, Tm, was determined by statistical analysis. The LPT and Tm were also assessed in fresh spermatozoa and spermatozoa incubated with egg yolk or egg-phosphatidylcholine (EPC) liposomes at 16°C, 12°C, 4°C, and 26°C (control). The results show that the membrane integrity of spermatozoa incubated at 16°C, 12°C, and 4°C decreased by 39%, 62%, and 67%, respectively, compared to the control. The LPT temperatures were between room temperature (26°C) and 10°C, with Tm at 14–16°C. The Tm for sperm incubated with liposomes or egg-yolk extender was below the measured range (2°C). Chilling sensitivity was found at a wide range of temperatures and transition temperatures, suggesting the presence of a wide variety of fatty acids (FAs) in the membrane with a high ratio of saturated-to-polyunsaturated FAs. Here we show that the protection afforded by the presence of egg yolk or liposomes in the extender is accomplished by shifting the Tm to below the 4°C point at which chilled semen is maintained for transport, or the point at which fast freezing begins to minimize cellular damage. Zoo Biol 0:1–13, 2005. © 2005 Wiley-Liss, Inc.
Article
Burgeoning application of oocyte cryopreservation in human assisted reproductive technologies (ART) has lead to several thousand births worldwide to date, and this in turn has begun to win the wider confidence of practitioners in the field of infertility therapy. Ethical and legal ramifications of embryo cryo-storage have prompted adoption of oocyte cryopreservation as a logical alternative. Facilitation of donor oocyte therapy via donor egg-banking is now being realized, and this will more than likely become the norm in the industry allowing appropriate quarantine, plus more cost-effective use for sharing of oocytes from each individual donor oocyte retrieval. Controversy still surrounds the practice of oocyte cryopreservation for women who wish to cryopreserve their oocytes at a younger age, for use later in life. However, when such fertility preservation is used prior to fertility-threatening disease therapy, then objections are mostly overturned in light of the growing positive experience with oocyte cryopreservation. Conventional slow-freezing of human oocytes has now been largely supplanted by the more predictable vitrification technology.
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The objective of the present work was to investigate the effect of dibasic sodium phosphate solution treatment on reproductive performance of unestrum Egyptian buffaloheifers. The study was conducted during the season of highest breeding activity of buffaloes in Egypt (October- March) on 60 anestric buffallo-heifers reared at Upper Egypt, Assiut province. All heifers were examined using an ultrasonic device 6/8 changeable linear probe once weekly. The heifers were divided randomly into two groups. The dibasic sodium phosphate-treated group (DHP, n=30) was injected with sterile anhydrous disodium hydrogen phosphate. The control group (C, n=30) was injected with saline s/c. Ovarian structures, follicles, corpora lutea and ovulation were recorded. Blood samples were collected twice per week for progesterone nalysis. Pregnancy diagnosis was determined at 40-50 days post service. The results showed that only 15 heifers had small follicles, while 45 heifers showed no ovarian structures. The number of heifers with functional ovaries did not increased in any group after treatment. The number of small sized follicles tended to be higher in (DHP) than in (C) (P = 0.06). There were no significant differences in serum progesterone concentration between groups (DHP: 0.70 ± 0.57 ng/ml versus C: 0.74 ± 0.62 ng/ml), the number of animals showing estrous signs (P = 0.69), time of appearance time of appearance and conception rate (P = 0.40). In conclusion, treatment of ovarian inactivity in buffalo-heifers with dibasic sodium phosphate tended to increase the number of small sized follicles but did not affect follicular growth, ovulation and consequently the resulting conception rate.
Article
Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All Fresh and cryopreserved oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group),Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos.
Article
Background: The cooling of mammalian oocytes to sub-physiological temperatures is widely known to affect their viability through the induction of various abnormalities at all stages of meiosis. Objective: This study was to compare the kinetics of nuclear status and oocyte damage in porcine, bovine and feline ovaries stored at 4 degrees C for 5 days. Methods: The nuclear status and oocyte quality during storage were evaluated before and after maturation culture. Results: The cold storage of ovaries decreased the proportions of porcine and bovine oocytes that remained at the germinal vesicle stage before maturation culture. The maturation rates of oocytes decreased with increasing storage time, independent of species. None of the porcine oocytes reached metaphase II (MII) after 1 day of storage. In contrast, bovine and feline oocytes from ovaries that were stored for 2 days and 3 days reached MII. DNA fragmentation in porcine oocytes from ovaries stored for 1 day was significantly higher than that in bovine and feline oocytes. Conclusion: The maturation competency of oocytes after the cold storage of ovaries could be related to the meiotic resumption of oocytes during storage and the occurrence of DNA fragmentation in oocytes during maturation culture.
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There are few reports of cryopreservation and injuries in Macrobrachium amazonicum embryos. Thus, the aim of this study was to analyze the effects of cryoprotectants agents and cooling on stage VIII of this species. Fertilized eggs from ovigerous females were removed from the incubation chamber, then placed in 10 ml Falcon tubes with a cryoprotectant solution and saline-free calcium solution. Thus, the embryos underwent a cooling curve of 1°C per min until reaching 5°C, and then were stored for 2 h. The tubes containing the embryos were washed to remove the cryoprotectant, acclimated for 5 min and then transferred to 50 ml incubators. At the end of the 24-h period, living embryos from each tube were counted and tabulated. A pool of embryos was fixed with 4% formaldehyde and then subjected to histology using 3-mm thick sections and stained with haematoxylin/eosin. Another pool was used for biometric analysis in which length, width and volume were analyzed. The cryoprotectants agents used were: dimethylsulfoxide (DMSO), methyl alcohol, ethylene glycol at 1, 5 and 10% and sucrose (0.5 M). Variance analysis was performed followed by Tukey's honest significant difference (HSD) test at 5% significance level. DMSO cryoprotectant affected embryo survival the least with rates of 71.8, 36.2 and 0% for concentrations of 1, 5 and 10%, respectively. Ethylene glycol caused 100% mortality at all the concentrations used. It was not possible to observe the interference of cooling and cryoprotectants on embryonic structures in this study.
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Sperm and embryos cryopreservation is a commonly applied technique for several years. Recently authorized in France, vitrification tends to replace gradually the conventional technique of slow freezing, so upsetting the practices in the management of patients. It allows from now on the cryopreservation of oocytes and opens new perspectives in egg donation either still in fertility preservation. This review thus attempted to examine the contribution of vitrification in the freezing of oocytes and human embryos at various stages of development. If obviously vitrification appears as the current method of choice for the cryopreservation of oocytes as well as blastocystes, the results are less cut as regards embryos to early stages. No increase in adverse obstetric and perinatal outcomes in children conceived from vitrified oocytes or embryos is noted in the literature. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
Article
Background: The primary problems with porcine oocyte vitrification are their low viability and development; both need improvement. Objective: This study was designed to improve the survival and developmental rates in vitrified-warmed porcine oocytes. Materials and methods: Porcine oocytes matured in vitro were vitrified-warmed with Cryotop. Then the oocytes were supplemented with Q10 during recovery culture. Results: The survival rates immediately after warming were 92.9% by morphological inspection and 39.3% by fluorescein diacetate (FDA) assay. The group of recovery culture with Q10 (VC+Q10) showed significantly higher viability compared to the group of recovery culture without Q10 (VC+) analyzed by morphology and the FDA. The VC+Q10 group showed a low Bax/Bcl-xl ratio and a high expression of MAP3K12 and TGFB3 compared to the VC+. The cleavage rate did not differ in both groups but, blastocyst yield was higher in VC+Q10 than the VC+ group. Conclusion: Supplementation of Q10 during recovery culture led to a higher blastocyst yield by increasing survival rates and regulating mRNA expressions.
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To test the feasibility of using the Modified-Cut Standard Straw (M-CSS) method for the vitrification of immature mouse oocytes. Additionally, the effects of different vitrification methods on oocyte survival, cytoskeletal organization, the distribution of cortical granules (CGs) and apoptosis were compared.Immature mouse oocytes were vitrified-thawed using electron microscope grid or M-CSS method, and then cultured to MII stage. Oocyte development, cytoskeletal organization, CG distribution and the expression of apoptosis-related genes were evaluated. Rates of recovery (91.7% vs. 74.9%) and survival (89.0% vs. 62.6%) were significantly higher in M-CSS group than in EM grid group. The number of oocytes with normal chromosome alignment at the spindle and spindle morphology were similar in both groups. However, the actin cap was significantly degraded in EM grid groups (52.6 vs 35.1%, respectively). Additionally, abnormal release of cortical granules is highly occurred in EM grid groups (42.6 vs 32.7%, respectively). Pro apoptosis-related gene expression levels of Bax, Caspase-3 were expressed lower than control in MII stage oocytes derived from M-CSS group; Anti apoptosis-related genes, survivin and Hsf-1 were slightly increased. However, all genes expression was significantly increased in MII stage oocytes derived from EM grid groups. Vitrification reduces survival rate of immature mouse oocytes, alters cytoskeletal organization and CG distribution, and promotes apoptosis. However, these effects are less pronounced in vitrified oocytes generated by M-CSS than in those generated by EM grid method. Therefore, the novel M-CSS is a feasible approach for the cryopreservation of immature mouse oocytes.
The cryopreservation of sperm, embryos and, oocytes plays an important and increasing role in assisted reproduction, due to improvements of old, and introduction of new technologies. In parallel, concerns are increasing about the technical and biological safety of this procedures.Cryopreservation of embryos is an established tool in human assisted reproductive technologies, and the clinical application of oocyte and ovarian tissue cryopreservation is increasing worldwide. Vitrification is a cryopreservation technique that leads to a glass-like solidification, with rapid cooling of cells or tissues. Today vitrification is seen as the future of cryopreservation of human oocytes and embryos, owing to improved survival rates and clinical outcomes.The aim of this study is to investigate the hypothetical risk of disease transmission through LN2 during the vitrification procedure if the cells are directly plunged into accidentally contaminated LN2. The need to guarantee the absolute sterility of LN2 for vitrification purposes is considered a critical problem.In this study we used different kind of microbes to infect group of mice oocyte stored in the same LN2 tank. The results of this study demonstrate absence of cross infection between samples during the cryopreservation and vitrification procedures. Although the microbes have grown in different kind of media such as embryo culture media or nutrient agar media to provide ideal environment for bacterial growing and more even the vitrification seems cause degeneration microbes. However, Safe and successful cryopreservation of oocytes requires screening of patients for infectious diseases, and application of the appropriate sanitary and cryoprocedures to ensure high postthaw survival of gametes and to minimize the risk of disease transmission to recipients.
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Oocyte cryopreservation is a promising option for long-term preservation of female genetics and fertility. It expands the usage of assisted reproductive technologies (ART) by producing embryo in vitro, exploits the genetic potential of superior females even after death, and avoids the ethical and legal controversies surrounding the preservation of human embryos. Oocyte cryopreservation is an important tool of fertility preservation in young women undergoing chemo- or radiotherapy for cancer treatment and those who plan to delay their child-bearing due to social reasons. The permeating cryoprotectants permeate the plasma membrane of cells and are generally considered cytotoxic. The addition of nonpermeating cryoprotectants in the medium counterbalances the osmotic shock and reduces the required concentration of permeating cryoprotectants ultimately decreasing their cytotoxic effect especially in vitrification method of cryopreservation. Cryotolerance of mammalian oocytes is different from sperm and embryos due to their unique morphological features.
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Embryo cryopreservation techniques have developed in order to preserve embryos obtained from superovulated cows. Research has followed trying to cryopreserve embryos and oocytes manipulated under in vitro conditions due to the low survival rates obtained with traditional cryopreservation methods. The different methodologies are discussed in the present review.
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PURPOSE: to verify vitrification techniques using 6 M DMSO to cryopreserve in vitro matured bovine oocytes, and to assess the effects of the time of exposure to vitrification solutions (VS). METHODS: dilutions of VS were prepared from the stock VS (VS 100%) consisting of 6 M DMSO to give 25 and 65% DMSO solutions. Bovine oocytes were in vitro matured for 18-22 h. Matured oocytes were placed first into 25% VS, at room temperature for 5 min, then transferred to 65% VS, before being pipetted into the 100% VS in plastic straws. Three experimental groups were formed: in the first group, time of pipetting through 65% VS and loading the straw took up to 60 s, in the second group it did not exceed 30 s. For thawing, straws were held in air for 10 s and then in a water bath for 10 s. The contents of each straw were expelled in sucrose solution and held for 5 min. In the third experimental group, oocytes went through all VS, but were not vitrified. All retrieved oocytes were inseminated. For control, fresh, in vitro matured oocytes were inseminated. RESULTS: after vitrification, 69.1 and 59.8% of the oocytes were retrieved from the 30 s and 60 s groups, respectively, and 93 and 89% of these oocytes appeared morphologically normal 24 h after insemination, respectively. In the group of oocytes exposed without vitrification, 75.6% were retrieved and 84.7% were morphologically viable, 24 h after insemination. No fertilization was observed in the experimental groups. Among controls, 65.4% were fertilized. CONCLUSIONS: the vitrification technique using 6 M DMSO is not a feasible approach to cryopreserve in vitro matured bovine oocytes. Decreasing the time of exposure to VS did not overcome deleterious effects of the procedure on the fertilizability of oocytes. Improvements in the technique are needed to protect the zona pellucida and oolemma.
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Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.
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Rational design of a cryopreservation protocol was demonstrated by using theoretical models of the cryopreservation process to develop an optimal freezing protocol for mouse oocytes. A coupled mechanistic model of the processes of freeze-induced cell dehydration and intracellular ice formation was developed, and cryomicroscopical measurements of intracellular ice formation kinetics were used to determine biophysical parameters required by the model, and to test model predictions of the freezing behaviour of mouse oocytes. A simple phenomenological model for oocyte damage resulting from exposure to concentrated electrolyte and cryoprotectant solutions during cryopreservation was obtained by defining a cost function equal to the duration of the freezing protocol. A two-step freezing protocol was theoretically optimized by using a sequential simplex algorithm to minimize the cost function, subject to the constraint that the predicted probability of intracellular ice formation remain below 5%, yielding a putative optimum at the cooling rate B = 0.59 degrees C/min, and plunge temperature Tp = -67 degrees C. By systematically varying B and Tp about these values in experiments with mouse oocytes cryopreserved in 1.5 M dimethyl sulphoxide, the maximal recovery of intact oocytes with a normal morphology (82%) was obtained for B = 0.5 degrees C/min and Tp = -80 degrees C. Further evaluation of the fertilizability and developmental capacity of oocytes cryopreserved using the optimized protocol yielded cleavage to the 2-cell stage in 65% of oocytes inseminated, and blastocyst formation in 50% of these 2-cell embryos.
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The ability to cryopreserve mammalian oocytes effectively would greatly increase their availability for a broad range of reproductive technologies. Oocytes have been frozen using both equilibrium and non-equilibrium approaches originally developed for mammalian cleavage-stage embryos, but rates of fertilization and development are typically much lower than with unfrozen oocytes. Production of live young from frozen oocytes of domestic animals has not been reported. Sensitivity of cytoskeletal elements, the meiotic spindle and other components of the cortical ooplasm to chilling and cryoprotective agents may contribute to the limited success in oocyte cryopreservation. Oocytes are also subject to physical events during freezing which influence cell survival. Estimates of biophysical parameters which influence the osmotic and cryobiological responses of oocytes are becoming available and may be useful for developing freezing protocols.
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The effects of intracellular lipid polarization and lipid removal treatments on the postthawedin vitrodevelopment of frozen bovine embryos at the 8-cell stage were studied. As the first step, bovine presumptive zygotes were centrifuged at 16,000gfor 20 min for the cytoplasmic lipid polarization and their lipid layers were removed by micromanipulation in order to examine the influence of these treatments on the developmental capacity of bovine zygotes. As the second step, bovine embryos developed to the 8-cell stage following centrifugation treatment at various forces (8000, 12,000, and 16,000g) or lipid removal treatment at the 1-cell stage were frozen in 1.8 M ethylene glycol + 0.05 M trehalose supplemented with 5% polyvinylpyrrolidone in a one-step procedure. There were no significant differences among the control (nontreatment), lipid-polarized, and lipid-removed groups with respect to the developmental capacity of fresh nonfrozen zygotes (experiment 1). The rates of survival and development to the blastocyst of frozen-thawed 8-cell embryos increased slightly with increasing force of centrifugation (experiment 2). The rate of development into blastocysts of the frozen-thawed 8-cell embryos was significantly higher in the groups that underwent centrifugation (at 16,000gfor 20 min;P< 0.05) or lipid removal (P< 0.01) treatments than the control (intact) group. However, there were no significant differences among the groups with respect to the rate of development to the expanded/hatched blastocyst stage. In addition, the mean cell numbers of embryos developed into blastocysts (day 8) derived from frozen-thawed 8-cell embryos tended to be low in the centrifugation and lipid removal groups compared to the controls (experiment 3). These results suggest that although the centrifugation with or without lipid removal treatments has no detrimental effects on the developmental capacity of bovine zygotes, the freezing tolerance of bovine 8-cell embryos was not improved by these treatments.
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The preservation of mammalian germ plasm by freezing has become an integral part of animal breeding, medicine, agriculture, reproductive biology and embryology. Considerable understanding of the physical‐chemical and physiological phenomena involved in cryopreservation of sperm, eggs and embryos has been achieved. This understanding has resulted in substantial improvements in the efficiency and efficacy of methods used to cryopreserve germ plasm. In addition, many of these methods have become integrated directly into the practice of animal breeding, and have contributed directly to the international trade in animal genetics. Development of these methods has been derived from close cooperation and interaction between the research and industrial communities. As the powerful techniques of molecular biology are focused on fundamental and applied aspects of embryology and reproductive biology, there are new problems regarding the cryobiology of germ cells to be solved.
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Bovine oocytes are damaged when chilled to temperatures near 0°C. We have determined the temperatures at which this injury occurs, as well as its kinetics and the functional consequences for oocytes both at the germinal vesicle-stage (GV) and after in vitro maturation (IVM). Cooling GV oocytes had no effect on their nuclear maturation or fertilization. Compared to control oocytes held at 30°C, the development of GV oocytes into blastocysts following maturation and fertilization was unaffected by cooling them to 20°C for 30 min (blastocyst formation: 25% vs 26%, respectively), but development decreased after cooling them to 10° and 0°C (blastocyst: 6% and 1%, respectively). Cooling oocytes after maturation gave similar results, with no difference between controls and oocytes cooled to 20°C (blastocyst: 25% and 26%, respectively). However, cooling them to 10° and O°C did reduce development (blastocyst: 8% and 3%, respectively). Chilling oocytes to 0°C for 30.sec reduced their cleavage and blastocyst formation by >40%; there was a high negative correlation between the length of exposure and subsequent survival, both for GV-stage and for IVM oocytes. The extreme sensitivity of both GV and IVM oocytes to chilling can explain the limited success obtained for cryopreservation of bovine oocytes by conventional slow-cooling procedures. © 1996 Wiley-Liss, Inc.
Article
The effects of cooling and warming on meiotic spindles of mouse oocytes have been assessed by transmission electron microscopy. Intact cumulus—oocyte complexes were immediately cooled from 37 to 15, 4, 0, and –7C (seeding temperature) for 15 min in a programmed biological freezer and fixed at these temperatures. Other complexes, cooled to these temperatures, were rapidly warmed to 37C and incubated for 2 hr before fixation at 37C. Of 334 oocytes assessed at various temperatures, at least 100 were examined for metaphase II spindles. Spindle microtubules completely disappear at 0 and –7C, while complete or partial depolymerization of microtubules was observed at 4C. Cooling to 15C did not cause major disruptions of spindle structure in most oocytes. Chromosomes tended to rotate or clump at lower temperatures but chromosome scatter outside the spindle zone was rarely observed. Centrosomal material was fragmented at 4C and occasionally at 15C and was not evident at the spindle poles at 0 and –7C. Kinetochores were seen at all temperatures. Spindle structure was evidently restored in the majority of oocytes on rewarming at 37C. Changes in the ooplasm induced by cooling were elongation and disruption of vesicular smooth endoplasmic reticulum, especially between lipid globules and disappearance of fibrillar inclusions. Cortical granule exocytosis was not observed on cooling, while microfilaments were intact. Swelling of membranous organelles was also observed in cumulus cells. Most of the cytoplasmic changes were also reversed on rewarming. The response of mouse oocytes to cooling is compared to that of human oocytes, reported previously.
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Thousands of live young of domestic animals have been produced by the transfer of cryopreserved embryos, indicating that such embryos can withstand exposure to extreme subzero temperatures. Paradoxically, some types of embryos may be severely altered or damaged simply by being cooled below physiological temperatures. For example, in vivo-derived embryos of pigs and in vitro-derived embryos of cattle are extremely sensitive to chilling below +14°C, whereas in vivo-derived embryos of cattle and sheep are not. This chilling sensitivity is dependent on the embryo's stage of development, and on the conditions under which it develops. Furthermore, an embryo's chilling sensitivity appears to be correlated with its freezing sensitivity. Understanding the basis of chilling injury will undoubtedly lead to a better understanding of freezing sensitivity, as well as of early embryology of domestic animals.
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The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane. In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown. On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.
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SUMMARY Three experiments were conducted to characterize the follicular oocytes of bovine ovaries and to assess the oocyte's ability to mature in vitro. Experiment 1 was designed to describe the oocytes from surface follicles of early luteal, luteal or follicular stage ovaries. Oocytes were also categorized on the basis of follicle size (1 to 3 or > 3 mm). Three classifi- cation schemes with numerical values assigned were developed to characterize investments of the oocyte, state of the ooplasm and stage of nuclear development. Characteristics typical of oocytes from nonatretic, vesicular follicles were given values of one. Characteristics with higher values were considered to be signs of degenera- tion. Oocytes from nine co~s per stage were evaluated. A higher proportion of nondegener- ate investments was found in follicles 1 to 3 mm in size (P
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The effect on the microtubule system of human oocytes of cooling to room temperature for either 10 or 30 minutes has been investigated. Changes in spindle organization were found in all oocytes cooled for 30 minutes compared with control oocytes kept at 37 degrees C throughout. These changes included reduction in spindle size, disorganization of microtubules within the spindle itself, and sometimes a complete lack of microtubules. In some oocytes, chromosome dispersal from the metaphase plate was associated with these changes. Cooling the oocyte to room temperature for only 10 minutes produced a similar pattern of disruption to spindle structure in many cases. The spindles in oocytes that were cooled for either 10 or 30 minutes and then allowed to recover at 37 degrees C for either 1 or 4 hours were found to resemble those in noncooled control oocytes in less than one half of the cases examined, although in only a few cases did the chromosomes remain dispersed. The significance of these findings for the handling of oocytes during gamete intrafallopian transfer and in vitro fertilization procedures is discussed in relation to the levels of aneuploidy detected in early human embryos.
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There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets is efficient; in a blind sorting experiment using freshly isolated islets and islets killed by either heat or alcohol, viability determined by insulin secretion in response to glucose stimulation correlated well with viability as determined by FDA/EB staining. Furthermore, it is possible to discriminate degrees of viability, and a scoring system is described for this purpose which is shown to correlate with another index of viability, the ATP content. A reliable viability stain should not itself be toxic; FDA/EB stained islets remain viable after staining, showing normal response to glucose stimulation and normal function after transplantation.
Article
Preovulatory human oocytes were cooled to 0 degrees C at 1 degree C/min, with or without the cryoprotectant dimethyl sulphoxide (DMSO), to assess the effects of cooling on the meiotic spindles and on oocyte structure. Batches of oocytes, cultured for 3-9 h, were held at 0 degrees C for 20 or 60 min and then fixed for transmission electron microscopy (TEM) either at 0 or 8 degrees C. Control oocytes were not cooled and were fixed at 22 or 37 degrees C for comparison. TEM revealed that 80% of the oocytes were at metaphase II, while 20% were at metaphase I and most had resumed meiosis recently. Control oocytes had more or less barrel-shaped meiotic spindles composed of microtubules (MT), some associated with chromosomes at kinetochores. Both metaphase I and II spindles were disassembled when cooled and fixed at 0 degrees C, with or without DMSO, due to extensive depolymerization of MT. The few MT that survived were found at the poles or were bundled together or were associated with chromosomes. Kinetochores were not prominent. Some oocytes cooled with DMSO and fixed at 0 or 8 degrees C showed evidence of MT, but the spindles were still disorganized and were abnormal in structure. Chromosomes tended to clump together or were dislocated in the cortical ooplasm in cooled oocytes, but widespread scattering was not observed. This was particularly evident in the absence of DMSO. Elements of the endoplasmic reticulum, Golgi, mitochondria and the cytosol were also adversely affected in some of the cooled oocytes and their surrounding cumulus cells. The results show that meiotic spindles are very sensitive to simple cooling and that DMSO does not provide substantial stabilization of the meiotic spindle even at 0 degrees C. The findings are discussed with reference to recent work on frozen human and mouse oocytes.
Article
Mouse oocytes were exposed to a variety of cooling regimes prior to insemination in vitro. Exposure to 4 degrees C, but not to 25 degrees C, was associated with a reduced fertilization rate, but development to the blastocyst stage of those oocytes that fertilized was not consistently different from that of non-cooled controls. The reduced fertilization rate seems to result from an effect of cooling on the zona pellucida, since it was not observed if the zona was removed prior to insemination, and since cooling rendered the zona pellucida resistant to the action of chymotrypsin. Using chymotrypsin resistance as an assay, the nature of the cooling-induced effect on the zona was investigated. It is suggested that rapid cooling to 4 degrees C may promote release of cortical granules and a premature zona reaction.
Article
In vivo developmental potentials of in vivo and in vitro matured oocytes fertilized in vitro were assessed in cattle. One-cell stages produced from in vivo matured oocytes developed into a pregnancy when transferred to the ampulla part of oviducts of synchronized heifers. In vitro matured oocytes achieved high penetration and cleavage rates but did not develop into pregnancies when transferred to synchronized heifers.
Article
The susceptibility of sheep oocytes to temperature changes during maturation in vitro was tested by reducing the incubation temperature to 20 degrees C at various stages of meiosis. Cooling induced chromosomal abnormalities including disorganized metaphase plates and multipolar spindles in 28-54% of oocytes cooled at all stages of meiosis from germinal vesicle breakdown (GVBD) to metaphase II. The time of GVBD (8-11 h after the start of culture) was the most sensitive to cooling, whereas fewest abnormalities were found in oocytes cooled in late metaphase I (16-19 h). In addition to the chromosomal abnormalities, unusual vesicles appeared in the cytoplasm of oocytes cooled at 8-11 h and 12-15 h. No abnormalities in protein synthesis were detected by one-dimensional SDS gel electrophoresis. The consequences of the abnormalities for the developmental potential of the cooled oocytes were tested by transfer to recipient ewes and fertilization in vivo. After 12 days of development only 6% and 11% oocytes cooled at 12-15 h and 20-23 h respectively had developed to expanded blastocysts, compared with 44% of control oocytes. The results demonstrated that maturing sheep oocytes are very sensitive to a drop in temperature.
Article
An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non-lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the non-lamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to non-lamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.
Article
Porcine embryos are cryopreserved using a method for reducing the level of lipid within cells of the embryo followed by freezing the embryo. Reduction of the level of lipid can be achieved either by polarizing the lipid within the embryo by centrifugation of the embryo, followed by rapid freezing or by microsurgical removal of the polarized lipid. Alternatively the level of lipid may be reduced by culturing the embryo in vitro. The reduction of the level of lipid before freezing results in good survival rates and can be used on zona intact porcine embryos.
Article
This study examined the effects of cryopreservation on cellular organization, chromosomal complement, and developmental potential of immature and mature mouse and human oocytes. Chromosomal analyses were performed by DNA fluorescence microscopy and karyotyping on the same metaphase II-stage oocytes before and after freezing. Cellular analyses involved electron microscopy, time-lapse video recording, and fluorescent-probe microscopy of cortical granules. The findings demonstrate that while profound cytoplasmic, nuclear, and nucleolar alterations occur in the immature oocyte during cryopreservation, an apparently normal nucleus and cytoplasm is re-established progressively after thawing and culture. The resulting oocytes mature at high frequency and for the mouse, are fertilizable and capable of normal preimplantation of embryogenesis. Cryopreservation of mature mouse and human oocytes is not accompanied by a significant increase in the frequency of aneuploidy. However, cryopreserved human oocytes, while fertilizable, arrest development during the early cleavage stages and display aberrant patterns of cytokinesis. The possible etiologies of developmental failure in the human embryo that may be related to oocyte cryopreservation, as well as the potential benefits of cryopreservation of the immature oocyte, are discussed with respect to clinical and commercial applications.
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Proteins belonging to a family of compounds known as "antifreeze proteins" interact with oocytes and protect the oolemma from damage at cryogenic temperatures. Experiments were performed with pig oocytes rapidly cooled to cryogenic temperatures in vitrifying solutions with and without antifreeze proteins. Four different types of antifreeze polypeptides and glycoproteins were tested. The integrity of the oolemma was examined with Fluorescein Diacetate (FDA) staining and morphological examinations. Results show that the pig oocyte oolemma is a primary site of injury during exposure to low temperatures and that all the different proteins have a similar ability to interact with and protect the oolemma. Our results may be important in developing solutions for long-term preservation of oocytes at cryogenic temperatures (cryopreservation).
Article
The purpose of this study was to evaluate effects of cooling and rewarming on the meiotic spindle apparatus of bovine oocytes. In experiment 1, in vitro-matured bovine oocytes were either maintained at 39 degrees C or cooled abruptly to 4 degrees C or approximately 25 degrees C. Immunohistochemical and DNA staining for visualization of microtubules and chromosomes, respectively, revealed an anastral, barrel-shaped spindle in bovine oocytes. Exposure to 4 degrees C for 10-20 min caused complete disappearance of the spindle. Some chromosome dispersion occurred after 60 min at 4 degrees C. After exposure to approximately 25 degrees C for 30 min, 90% of oocytes appeared abnormal, having either an abnormal spindle or no spindle. In experiment 2, oocytes cooled to either approximately 25 degrees C or 4 degrees C for 30 min were rewarmed directly or in steps for 15 or 60 min. Spindles did not return to normal in most oocytes regardless of cooling temperature or rewarming scheme. Step-wise rewarming was no more beneficial than direct rewarming. More of the oocytes rewarmed directly contained dispersed chromosomes as time at 39 degrees C increased.
Article
When cells are cooled to temperatures above the freezing point of water at rates greater than a few degrees per minute, they sustain irreversible injury. Reduction of this "cold shock" damage could increase the survival of animals and plants at low environmental temperatures and improve the cryopreservation of plant and animal cells. Leakage of solutes across membranes, associated with thermotropic phase transitions in membrane lipids, is thought to be responsible, but this hypothesis has not been tested directly. Using Fourier transform infrared spectroscopy (FTIR), we measured the lipid phase transitions in intact, living sperm, the animal cell in which cold shock has been studied most extensively. A shift in the CH2 absorbance peaks indicates the transition from liquid-crystalline to gel phase. The phase transition in sperm membranes occurred at a lower temperature for a marine shrimp than for the pig. In each case, potassium leakage, which is a hallmark of cold shock damage, increased abruptly near the end of the phase transition. Human sperm are quite resistant to cold shock, and an abrupt lipid phase transition was not detected. This phase behavior is typical of membranes containing a high proportion of cholesterol, and human sperm have an unusually high sterol content. High cholesterol levels are known to stabilize membranes during cooling. Overall, the lipid phase behavior was consistent with the temperature range over which cooling was damaging for pig and shrimp sperm, and the with the extent of damage produced in pig and human sperm. This is the first direct evidence that cold shock results from lipid phase transitions in cell membranes.
Article
A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between DCI in sperm and the thermotropic phase transition of their membrane lipids was demonstrated. In the present study, we examined the phase transition of the membrane lipids of immature and in vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20 degrees C, while a very broad phase transition, which centered around 10 degrees C, was observed for mature oocytes (MII) stage. Thermotropic phase transitions were demonstrated to be related to the temperature at which DCI affected the integrity of the oocyte membranes. When immature oocytes were cooled to 13 degrees C, fewer oocytes (40%) retained their membrane integrity than after exposure to 4 degrees C (51%) or holding them at 38 degrees C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differences in membrane integrity were observed when in vitro-matured oocytes were cooled to the same temperatures. Subsequently, GV oocytes were cooled to 4 degrees C, and 26% underwent maturation and 19% underwent fertilization in vitro. In vitromatured oocytes that were cooled to 4 degrees C displayed a slightly decreased rate of fertilization; the overall fertilization was 60% with 24% polyspermy, rather than the 76% fertilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes. Nucleated bovine GV oocytes were electrofused with in vitro-matured and enucleated oocytes, and then cooled to 4 degrees C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibility to low temperatures. This finding led to an improvement in the survival of oocytes after cryopreservation.
Article
In vitro matured bovine oocytes were frozen slowly in 1.6 M 1,2-propanediol following centrifugation treatment for polarization of lipid droplets in the cytoplasm. After thawing, the survival of the oocytes was assessed morphologically and also by in vitro fertilization and culture. The polarization of cytoplasmic lipid droplets had a negative effect on the survival of frozen-thawed oocytes. Thus, this treatment did not improve the frequency of normal fertilization and development to blastocysts, compared with that of frozen control oocytes. However, the frequency of polyspermy and activation of lipid-polarized oocytes that survived after freezing-thawing and subsequent in vitro fertilization tended to be less than those of surviving control oocytes. In addition, the effect of centrifugation treatment was to produce a small but significant increase in the cleavage rate of oocytes that survived after freezing-thawing and the development rates to blastocysts of surviving lipid-polarized oocytes tended to increase, compared with those of surviving control oocytes. These results suggest that the freezing tolerance of the spindle and other organelles of in vitro matured bovine oocytes is associated with lipid droplets and may be improved by the polarization of cytoplasmic lipid droplets before cryopreservation.
Article
Effective use of encapsulated sperm requires careful review of: (a) the conditions under which the procedure can be effectively used; (b) assessment of the effect of storage conditions on sperm survival; (c) description of the environment of the female tract before, during and after capsule deposition; and (d) economic evaluation of impact and costs of the putative technology. Sperm survival depends on successful sustenance over two periods of storage (at subambient temperatures after collection and extension, then at body temperature when placed in the female tract) and one period of action (after release and until fertilization). The bioenergetic requirements of cauda epididymal and ejaculated bull and ram sperm are reviewed in terms of absolute ATP needs and are discussed in terms of storage needs. In addition, sperm inactivation by lipid peroxidation is discussed and suggestions are provided to minimize the process. Two general types of containers are possible. An open porous form allows free passage of nutrients and metabolic products; the entrapped sperm are thus subjected to the changing environment in the female tract. The other form is a sealed capsule that opens to release sperm before ovulation; it provides a sperm storage environment independent of female tract chemistry but introduces problems of nutrient supply and metabolite release. Potential experimental approaches to evaluate each type of system are discussed.
Article
Oocytes have been successfully cryopreserved using rapid and slow freezing procedures. However, variability in the success of replicates has limited its practical application. In the present study, mature mouse oocytes were vitrified in 6 M dimethyl sulfoxide supplemented with 1 mg/ml antifreeze glycoproteins (AFGP) (solution known as VSD + AFGP) from the blood of Antarctic notothenioid fish. Such AFGPs have been used to protect mammalian cells during hypothermia and cryopreservation. However, the degree of protection afforded is a contentious issue. Stepwise addition of cryoprotectant was performed either at room temperature (19-21 degreesC) or on ice (2-4 degreesC), at the final stage of which oocytes were pipetted into 0.25 ml plastic insemination straws and held in liquid nitrogen vapor at -140 degreesC for 3 min before being plunged into liquid nitrogen. Thawing involved holding the straw in the air for 10 s and then in water at 20 degreesC for 10 s before dilution of the VSD solution with 1 M sucrose. Viability was assessed by in vitro fertilization; results have been quoted as median (range). Statistical analyses were performed using Kruskall-Wallis and Mann-Whitney U tests (P < 0.05). Of the oocytes cryopreserved following exposure to VSD + AFGP at room temperature (n = 518, 15 experimental runs), 78% (0-94%) retained normal morphology and, of these, 53% (0-100%) cleaved to two cells. Of these two-cell embryos, 56% (0-100%) went on to develop to blastocyst. The overall percentage development to blastocyst, i.e., number of blastocysts/total number of oocytes treated x 100, was 20% (0-76%). Exposure of oocytes to the VSD + AFGP on ice prior to cryopreservation yielded significantly improved rates of fertilization (94%, 82-100%) and overall development to blastocyst (66%, 24-89%) when compared with oocytes cryopreserved following exposure to the VSD + AFGP at room temperature. Rates of normality (86%, 35-95%) and development to blastocyst (89%, 64-100%) were also improved. Cryopreservation in 6 M dimethyl sulfoxide supplemented with 1 mg/ml AFGP resulted in poor rates of survival which were highly variable when exposure to cryoprotective agent (CPA) was performed at room temperature. Lowering the temperature of exposure to CPA prior to cryopreservation resulted in improved viability.
Article
The present study was conducted to clarify the effect of heparin dosage and sperm capacitation time on in vitro fertilization (Experiment 1) and cleavage (Experiment 2) rates of bovine oocytes matured in vitro. For in vitro fertilization, seven dosages of heparin (0, 5, 10, 25, 50, 100 and 200 microg/ml) and nine incubation periods (0, 5, 15, 30, 45, 60, 120, 180 and 240 min) in a capacitation medium were examined, using 6,634 oocytes. The mean proportions of fertilized oocytes in 25, 50 and 100 microg/ml of heparin were significantly (P<0.05) higher (53 to 59%) than in the other dosages (3 to 44%). Incubation with heparin for longer than 60 min lowered the frequencies of fertilization (20 to 36%) compared with the shorter incubation periods (38 to 49%). Higher proportions of fertilized oocytes were obtained by 5, 15, 30 or 45 min of incubation (42 to 49%) than by the other time periods (20 to 38%). Cleavage rates were found by using 2,098 oocytes in a factorial study (4x4x15: dosages -25, 50, 100 and 200 mug/ml; incubation periods -0, 15, 30 and 60 min; and replicates). The incubation periods and replicates resulted in highly significant differences (P<0.001) in development rates to eight-cell stage, but the four dosages of heparin showed no significant differences. The present results indicate that heparin dosage and sperm capacitation time are important factors influencing in vitro fertilization and cleavage rates. Optimal heparin dosages for the capacitation of bull spermatozoa ranged from 25 to 100 microg/ml; optimal incubation periods ranged from 5 to 60 min.
Article
Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.
Vitrification of oocytes and embryos New Trends in Embryo Transfer
  • A Arav
Arav, A. Vitrification of oocytes and embryos. In " New Trends in Embryo Transfer " (A. Lauria, and F. Gan-dolfi, Eds.) pp. 255–264.
Strategies to maximize selection progress in dairy cattle Genetics Applied to Livestock Production
  • J A Wooliams
Wooliams, J. A. Strategies to maximize selection progress in dairy cattle. In " Genetics Applied to Livestock Production. " Proc IV World Congress, pp. 15–24, 1990.
Cryoprotectant and thermal effects on cytoskeletal organization and IVF rate of mouse oocytes
  • Overstrom
Overstrom, E. W., Paquin-Platls, D., Toner, M., and Cravalho, E. G. Cryoprotectant and thermal effects on cytoskeletal organization and IVF rate of mouse oocytes. Biol. Reprod. 42 (Suppl. 1), 175 (1990).
Vitrification of oocytes and embryos
  • Arav
Effects of cooling and rewarming on the meiotic spindle and chromosomes ofin vitro
  • Aman
Vitrification of mature mouse oocytes in a 6 M Me2
  • O'Neil
Strategies to maximize selection progress in dairy cattle
  • Wooliams
Effect of heparin dosage and sperm capacitation time onin vitro
  • Fukui
In vivoin vitroin vivoin vitro
  • Greve
Effects of centrifugation and lipid removal on the cryopreservation ofin vitro
  • Murakami
The decreased membrane fluidity ofin vivo
  • Takeshi