Eva Mocé

Use of Cholesterol in Sperm Cryopreservation: Present Moment and Perspectives to Future

CONTENTS Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival

Acceptable fertility using cryopreserved ram sperm is currently only achieved using laparoscopic intrauterine insemination. Improving the cryosurvival of ram sperm may permit greater fertility rates using more practical techniques. This study was conducted to determine if treating ram sperm with six different cyclodextrins pre-loaded with cholesterol (CLC), prior to cryopreservation increases sperm cryosurvival and if this technology can be used with neat semen. Subsequent experiments evaluated how adding CLC to sperm affected sperm cholesterol content, sperm osmotic tolerance limits, sperm post-thaw survival after incubation and the capacity of sperm to bind to zona pellucidae of cattle and sheep oocytes. Sperm treated with 2-hydroxypropyl-beta-cyclodextrin prior to cryopreservation exhibited greater percentages of motile sperm (62%) compared to the control (no CLC treatment) samples (43%, P<0.05), after thawing. In addition, samples treated with methyl-beta-cyclodextrin exhibited percentages of motile and viable sperm similar to samples treated with 2-hydroxypropyl-beta-cyclodextrin. Other CLC-treated samples were similar to the control. The CLC concentration that optimized sperm cryosurvival was 2mg CLC/120 x 10(6) sperm for both methyl-beta- and 2-hydroxypropyl-beta-cyclodextrin when added to neat semen prior to cryopreservation. Addition of 2mg CLC not only maintained greater percentages of motile sperm compared to the control samples, but maintained greater percentages of motile sperm during a 3h incubation after thawing. In addition, 2-hydroxypropyl-beta-cyclodextrin pre-loaded with cholesterol maintained greater percentages of viable sperm (33%), than control sperm (18%; P<0.05). Treating ram sperm with CLC increased the sperm cholesterol content>1.9-fold and although some cholesterol was lost from the sperm during cooling and cryopreservation, the cholesterol content remained greater in CLC-treated sperm after cooling and after thawing than in control sperm (P<0.05). In addition, CLC-treated sperm maintained greater percentages of motile sperm through a wide range of osmotic solutions (150 and 425 mOsm) while control sperm lost motility in solutions outside a more narrow range (270 to 370 mOsm). Greater numbers of CLC-treated sperm bound to zona pellucida than control sperm (P<0.05), although number of sperm binding cattle and sheep oocytes, was similar (P>0.05). In conclusion, treating ram sperm with CLC increases sperm cryosurvival rates and sperm longevity after thawing. It also increases the cholesterol content, osmotic tolerance, and zona-binding capabilities of sperm. Finally, CLCs can be added to neat semen, making this technology feasible for practical application using current cryopreservation techniques for ram semen.

Rabbit sperm cryopreservation : A review

Sperm cryopreservation is a great challenge, since many sperm are irreversibly damaged or present altered functionality after the whole process. Although components of extenders for sperm cryopreservation are quite similar between species, sperm from each of the species present peculiarities that force researchers to optimize the extenders and protocols for each particular species. In this review, information related to rabbit sperm cryopreservation is compiled. The topics discussed include the extenders and protocols developed for rabbit sperm cryopreservation, as well as fertility data obtained after artificial insemination with cryopreserved sperm and factors that may have an impact on the results obtained. In addition, suggestions for improving the results after cryopreservation of rabbit sperm are also proposed.

Treating boar sperm with cholesterol-loaded cyclodextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability.

Treating sperm with cholesterol-loaded cyclodextrins (CLC) improves the cryosurvival of the sperm of different cold-shock sensitive species. However, the response of boar sperm to this treatment is not fully understood. The aim of this study was to determine how CLC and methyl-β-cyclodextrin (MβCD, not loaded with cholesterol) affect the parameters for boar sperm functionality, including sperm osmotic resistance, and the ability of the sperm to capacitate and to penetrate the sows immature oocytes in vitro. Samples treated with CLC or MβCD prior to freezing exhibited similar percentages of motile sperm, live sperm and sperm with intact acrosomes as the control samples (P>0.05). In addition, these treatments did not alter the response of the boar sperm to capacitating conditions. However, when compared to the controls and the MβCD-treated samples, the CLC-treated sperm maintained greater percentages of motile sperm and live sperm in a wide range of osmotic solutions including hypo- (50, 75 and 150 mOsm/kg) and hyper-osmotic (600, 800 mOsm/kg) conditions (P<0.05). In addition, the CLC-treated sperm exhibited greater oocyte penetration ability than the control and the MβCD-treated sperm (P<0.0001). In conclusion, the pre-freezing treatment of boar sperm with CLC does not alter the ability of the sperm to respond to capacitating conditions. Despite not increasing the cryosurvival of the sperm, this treatment widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability.

Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation

The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose-egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (P<0.05). However, from the three monosaccharides tested, glucose provided the best sperm quality after freezing-thawing. With respect to the disaccharides studied, samples frozen with the extender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (P<0.05) in the samples cryopreserved with the trehalose extender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition.

The fertility of ram sperm held for 24 h at 5°C prior to cryopreservation.

Diluted ram sperm can be held for 24h at 5 degrees C prior to cryopreservation without impacting cryosurvival rates, however, the effects this storage has on subsequent fertility are unknown. These studies were conducted to evaluate the fertility of semen held for 24h (to mimic shipping semen to a cryopreservation center), prior to freezing. Semen from Suffolk rams (n=3 in experiment 1 and n=6 in experiment 2) with initial motility of greater than 70%, were diluted to 200 x 10(6)sperm/mL, in one step, with a Tris-egg yolk-glycerol diluent. In experiment 1, diluted samples were cooled to 5 degrees C over 2h, and then divided. Sperm in one fraction were loaded into 0.5mL straws, frozen (T0) and stored in liquid nitrogen until thawing. Sperm in the second fraction were held at 5 degrees C for 24h (T24) before being frozen. In experiment 2 ejaculates were collected and divided into two fractions. Sperm in one fraction were treated with cholesterol-loaded cyclodextrin (CLC) and sperm in the other served as control. Both fractions were diluted, cooled, and cryopreserved as described in experiment 1. Stage of the estrous cycle was synchronized in ewes (n=196) using controlled internal drug releasing devices (CIDR) for 12d and at CIDR removal each ewe was administered PMSG (500IU in experiment 1 and 350IU in experiment 2) immediately before insemination. Ewes were stratified by age and randomly assigned to one of the semen treatments; experiment 1: Fresh (F), T0, or T24; experiment 2: F, T24, or CLC, and inseminated laparoscopically 56h after CIDR removal. Differences in fertility were detected between experiments, but not for treatments within experiments. Differences in fertility were also observed due to ewe age, with the 3-year-old ewes having the greatest fertility (50.7%) and 6-year-old ewes having the least fertility (9.6%; P<0.05). Differences in the prolificacy rates due to semen treatment were also observed but differences due to ewe age were not detected. Therefore, sperm can be held at 5 degrees C for 24h prior to cryopreservation without altering sperm fertility.

Response of Boar Sperm to the Treatment with Cholesterol-Loaded Cyclodextrins Added Prior to Cryopreservation

Cryopreserved boar sperm is not used extensively for artificial insemination, owing to the poor fertility rates of the sperm after freezing and thawing. The sperm membrane is damaged as the cells are cooled from body temperature to 5°C (cold shock), as well as during the freeze-thaw process. Increasing the cholesterol content of boar sperm membranes could help them survive cryopreservation, similar to sperm from other species that are cold shock sensitive. The aim of this study was to determine the optimal cholesterol-loaded cyclodextrin (CLC) concentration to use for boar sperm cryopreservation, and the influence of CLCs on the cryosurvival of sperm from boars classified as good or poor freezers. Treating boar sperm with 1 mg of CLC/120 × 10(6) sperm slightly improved (p < 0.05) the percentage of viable sperm after freezing-thawing. On the other hand, sperm, from both good and poor freezers, responded similarly to CLC treatment. Nevertheless, additional studies will be needed to study the effect of this treatment on other parameters of sperm quality.

Is Sperm Freezability Related to the Post-Thaw Lipid Peroxidation and the Formation of Reactive Oxygen Species in Boars?

The aim of the present study was to determine whether the levels of reactive oxygen species (ROS) substances production and the levels of lipid peroxidation of the sperm membrane were related to the quality that the ejaculates exhibited after cryopreservation in boars. Ejaculates from 42 healthy boars were used in this study and they were cryopreserved with the lactose-egg yolk extender (LEY). Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37 °C after thawing: the percentage of sperm with intact plasma membrane (SIPM), intracellular reactive oxygen substances production through mean of DCF fluorescence intensity of total sperm (mean-DCF) and the percentage of viable and non-viable sperm containing oxidized BODIPY (VSOB and NVSOB). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. The classification of the ejaculates into good or bad freezers was performed through hierarchical cluster analysis from SIPM and TMS at 150 min post-thawing. The ejaculates of those males classified as good freezers exhibited higher (p < 0.05) SPIM, TMS and PMS than the bad freezers, although both groups presented similar (p > 0.05) VSOB, NVSOB and mean-DCF. Therefore, these results show that lipid peroxidation and the amount of reactive oxygen substances in the sperm after cryopreservation are similar between boars classified as good or bad freezers.

In vivo fertilising ability of frozen-thawed boar sperm treated with cholesterol-loaded cyclodextrins prior to cryopreservation

The use of frozen-thawed (FT) sperm for the artificial insemination of pigs is rare. Treating boar sperm with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances the penetration of immature oocytes in vitro, and this phenomenon has been positively correlated with in vivo fertilisation ability in pigs. The objective of this study was to compare the in vivo fertilising ability of boar sperm treated with 0 (control) or 1mg CLC/120×10(6) sperm (CLC) prior to freezing. The fertilising ability of the FT sperm was compared in hormonally treated (equine/human chorionic gonadotropin hormones; eCG/hCG) weaned sows inseminated once (cervical insemination) at the following fixed-times after hCG administration: 37h (experiment 1) or 30h (experiment 2). In experiment 1, both treatments exhibited similar fertility rates of 67.7 and 55.9% for the control and CLC, respectively (P>0.05); however, the CLC group had a smaller litter size (11.3±0.9) than the control group (13.6±0.8) (P<0.05). In experiment 2, the pregnancy and farrowing rates were 65.2 and 66.7% for the control and CLC groups, respectively, and the litter size was 12.9±1 and 11.3±1 for the control and CLC groups, respectively, which were similar (P>0.05) for both treatments. These results indicate that the timing of insemination developed for the FT control sperm may not be suitable for CLC-treated sperm and that CLC-treated sperm may benefit from a shorter time interval between the hCG treatment and insemination. Moreover, acceptable results can be obtained with FT boar sperm with a single artificial insemination performed 30h after hCG treatment, which is the time interval recommended for fresh sperm.

Egg yolk and glycerol requirements for freezing boar spermatozoa treated with methyl β-cyclodextrin or cholesterol-loaded cyclodextrin.

Egg yolk (EY) and glycerol are common constituents of extenders used for sperm cryopreservation. It has been demonstrated that using cholesterol-loaded cyclodextrins (CLC) improves sperm cryosurvival in several species. However, standard freezing extenders might not be the most appropriate for CLC-treated sperm. This study evaluated the EY and glycerol requirements for freezing CLC-treated boar spermatozoa. Semen samples from 34 ejaculates coming from 4 boars were used. Each ejaculate was split into three aliquots: one was used untreated (control), and the other two were treated with 1 mg of CLC or methyl-β-cyclodextrin/120 × 106 sperm for 15 min at 22 C prior to cryopreservation. Our results indicated that reducing the concentration of EY was detrimental for sperm viability after thawing (31.57 ± 2 vs. 19.89% ± 2 for 20 and 10% EY, respectively; P <0.05), even in semen treated with CLC. On the other hand, it was observed that the traditional concentration of glycerol (3%) was not the appropriate for freezing CLC-treated sperm (61.10 ± 3 vs. 47.87% ± 3 viable sperm for control and CLC-treated sperm, respectively; P <0.05). Thus, CLC-treated sperm showed a higher tolerance to high glycerol concentrations (5%) in terms of sperm viability (59.19% ± 3) than non-treated sperm (45.58% ± 3; P<0.05). Therefore, it could be necessary to modify the freezing extenders for CLC-treated sperm. Nevertheless, additional studies will be needed to evaluate alternative cryoprotectants and to determine the effect of high glycerol concentrations on sperm functionality.