Effects of forskolin on cryopreservation and embryo development in the domestic cat.

One of the approaches to improve cryotolerance in lipid-rich embryos is to modify their lipidome in vitro. This work is aimed to study the effects of forskolin exposure on the in vitro embryo development of the domestic cat and to evaluate how the change in lipid content affects the cryopreservation results. In vitro-derived embryos were cultured with 10 µM forskolin from the 2-cell stage for 24 h or 96/168 h to the morula/blastocyst stage. Some of the embryos treated with forskolin for 24 h were cryopreserved with slow freezing, the other ones were used to characterize their developmental rates and the amount of intracellular lipids. The in vitro exposure to forskolin had a positive effect on the embryo development, as more embryos developed to the morula stage in the forskolin-treated group (92.9%) compared to the controls (64.7%) after 120 h of in vitro culture (IVC). Nile Red staining revealed a reduced amount of intracellular lipids in the forskolin-treated embryos. The percentage of embryos developed to the morula stage was lower in the frozen-thawed embryos not treated with forskolin (54.5%), but not in the frozen-thawed forskolin-treated group (63.6%) as compared to non-frozen controls (80.8%). Thus, the exposure of embryos to forskolin in vitro reduced the level of intracellular lipids and affected embryo development before and after cryopreservation.

Lipid phase transitions in cat oocytes supplemented with deuterated fatty acids.

Cryopreservation of oocytes has already been used to preserve genetic resources, but this technology faces limitations when applied to the species whose oocytes contain large amounts of cytoplasmic lipid droplets. Although cryoinjuries in such oocytes are usually associated with the lipid phase transition in lipid droplets, this phenomenon is still poorly understood. We applied Raman spectroscopy of deuterium-labeled lipids to investigate the freezing of lipid droplets inside cat oocytes. Lipid phase separation was detected in oocytes cryopreserved by slow-freezing protocol. For oocytes supplemented with stearic acid, we found that saturated lipids form the ordered phase being distributed at the periphery of lipid droplets. When an oocyte is warmed to physiological temperatures after cooling, a fraction of saturated lipids may remain in the ordered conformational state. The fractions of monounsaturated and polyunsaturated lipids redistribute to the core of lipid droplets. Monounsaturated lipids undergo the transition to the ordered conformational state below -10°C. Using deuterated fatty acids with a different number of double bonds, we reveal how different lipid fractions are involved in the lipid phase transition of a cytoplasmic lipid droplet and how they can affect cell survival. Raman spectroscopy of deuterated lipids has proven to be a promising tool for studying the lipid phase transitions and lipid redistributions inside single organelles within living cells.

Effects of slow freezing and vitrification on embryo development in domestic cat.

Cryopreservation of gametes and embryos is used to maintain genetic diversity of domestic and wild felids. However, felid oocytes and preimplantation embryos contain large amount of intracellular lipids, which affect their cryosensitivity. The objective was to compare the effects of slow freezing and vitrification and to study lipid phase transition (LPT) during cooling in cat embryos. In vitro-derived embryos were cultured 48 hr up to 4-8 cell stage, thereafter were either slow frozen or vitrified. Propylene glycol (PG) alone was used as a cryoprotective agent (CPA) for slow freezing, and a mixture of PG and dimethyl sulfoxide (DMSO) were used as CPAs for vitrification. After thawing/warming, embryos were in vitro cultured additionally for 72 hr. The total time of in vitro culture was 120 hr for all the groups including non-frozen controls. Effects of both cryopreservation procedures on the subsequent embryo development and nuclear fragmentation rate in embryonic cells were compared. There was no significant differences among the percentages of embryos achieved morula and early blastocyst stage in frozen-thawed group (36.4% and 20.0%), in vitrified-warmed group (34.3% and 28.6%) and in controls (55.6% and 25.9%). Cell numbers as well as nuclear fragmentation rate did not differ in these three groups. Average lipid phase transition (LPT) temperature (T*) was found to be relatively low (-2.2 ± 1.3°C) for the domestic cat embryos. It is supposed that the low LPT of LDs may provide a good background for successful application of slow freezing to domestic cat embryos. Generally, our study indicates that slow freezing and vitrification are both applicable for domestic cat embryo cryopreservation.

Influence of Cellular Lipids on Cryopreservation of Mammalian Oocytes and Preimplantation Embryos: A Review.

Lipids are among the most abundant and essential cell components. Specifically, cytoplasmic lipid droplets (LDs) play crucial roles in cellular energy homeostasis. The foci of this review are (1) the composition and roles of lipids during oocyte maturation and early embryonic development, (2) possible causes of cryoinjuries in lipid-rich oocytes/embryos, and (3) ways to overcome these detrimental effects. Recent reports show that LDs in oocytes and embryos are not only energy depots but also are active organelles, possessing many other functions. In addition, analysis of the current literature confirms that lipid phase transition followed by phase separation during cryopreservation is one of the major causes of cryodamage in lipid-rich oocytes and embryos. While LDs and cell membranes are sensitive to chilling and freezing conditions, recent advances in vitrification and delipidation of lipid-rich oocytes and embryos partly mitigate cryodamage. The multidisciplinary approach is critical to reveal mechanisms underlying cryodamage and provides a theoretical basis for optimal cryopreservation of lipid-rich oocytes/embryos.

Lipid Droplet Phase Transition in Freezing Cat Embryos and Oocytes Probed by Raman Spectroscopy.

Embryo and oocyte cryopreservation is a widely used technology for cryopreservation of genetic resources. One limitation of cryopreservation is the low tolerance to freezing observed for oocytes and embryos rich in lipid droplets. We apply Raman spectroscopy to investigate freezing of lipid droplets inside cumulus-oocyte complexes, mature oocytes, and early embryos of a domestic cat. Raman spectroscopy allows one to characterize the degree of lipid unsaturation, the lipid phase transition from the liquid-like disordered to solid-like ordered state, and the triglyceride polymorphic state. For all cells examined, the average degree of lipid unsaturation is estimated as ∼1.3 (with ±20% deviation) double bonds per acyl chain. The onset of the lipid phase transition occurs in a temperature range from -10 to +4°C and does not depend on the cell type. Lipid droplets in cumulus-oocyte complexes are found to undergo abrupt lipid crystallization shifted in temperature from the ordering of the lipid conformational state. In the case of mature oocytes and early embryos obtained in vitro, the lipid crystallization is broadened. In the frozen state, lipid droplets inside cumulus-oocyte complexes have a higher content of triglyceride polymorphic ß and ß' phases than estimated for mature oocytes and early embryos. For the first time, to our knowledge, the temperature evolution of the phase state of lipid droplets is examined. Raman spectroscopy is proved to be a promising tool for in situ monitoring of the lipid phase state in a single embryo/oocyte during its freezing.

Sperm cryopreservation in the Far-Eastern wildcat (Prionailurus bengalensis euptilurus).

The Far-Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far-Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far-Eastern wildcat captive-born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen-thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2% ± 3.0% for the domestic cat and 38.0% ± 10.2% for the Far-Eastern wildcat. The motility of frozen-thawed spermatozoa was 30.8% ± 9.8% for the domestic cat and 33.7% ± 15.1% for the Far-Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9 ± 5.9 for the domestic cat and 55.0% ± 6.4% for the Far-Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2% ± 4.8% and 30.8% ± 4.4% of all reported anomalies for the domestic cat and Far-Eastern wildcat, respectively). Domestic cat epididymal and Far-Eastern ejaculatory spermatozoa fertilized in vitro-matured oocytes of the domestic cat (30.0% ± 5.5% and 35.5% ± 15.0%, respectively). Taken together, these results suggest that the freezing of Far-Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.

Characteristics and fertility of domestic cat epididymal spermatozoa cryopreserved with two different freezing media.

The study represents a comparison of cryopreservation of domestic cat epididymal spermatozoa with two commercially available freezing media: CaniPlus Freeze (CPF) and SpermFreeze (SF). The viability of nonfrozen spermatozoa evaluated by the VitalScreen test was 68.7 ±â€¯3.0%. These figures were lower for the frozen-thawed spermatozoa: 51.2 ±â€¯6.3% for CPF group and 54.4 ±â€¯3.1% for SF group. The motility of nonfrozen spermatozoa was 57.2 ±â€¯4.5%. These figures were reduced in both frozen-thawed groups; however, there was no significant difference in these parameters between CPF (30.8 ±â€¯7.1%) and SF (27.4 ±â€¯8.1%) groups. The percentage of nonprogressively moving motile spermatozoa after freezing-thawing was decreased in both frozen-thawed groups (23.5 ±â€¯5.9 and 12.0 ±â€¯2.4 for CPF and SF frozen correspondingly) as compared with nonfrozen controls (42.1 ±â€¯4.1%). Morphology of spermatozoa was assessed by light microscopy. The mean percentages of normal spermatozoa were 28.5 ±â€¯4.1% for nonfrozen group, 26.0 ±â€¯2.3% for CPF frozen group, and 23.9 ±â€¯1.9% for SF frozen group. The most frequent anomalies in all the three groups were flagella and combined defects. In vitro fertilization (IVF) of domestic cat oocytes with nonfrozen and frozen-thawed spermatozoa produced developing embryos. The percentage of in-vitro-derived embryos was 43.6% after using nonfrozen spermatozoa. Frozen-thawed spermatozoa developed at a similar rate (44.0%) after using SF. However, the rate of embryo development was lower (20.1%) when CPF was used. The in-vitro-derived embryos in the nonfrozen group consisted of 46.9 ±â€¯2.5 cells after 5-day culturing. After cryopreservation with SF and CPF the cell numbers per embryo were 39.9 ±â€¯2.7 and 31.8 ±â€¯3.4 correspondingly. In CPF group these numbers were lower than in nonfrozen controls. Cryopreservation of spermatozoa with either of two freezing media led to a decrease in post-thaw viability and motility of spermatozoa but did not affect the rate or spectrum of their morphological anomalies. The use of CPF, but not SF led to a decrease of sperm fertilizing abilities.