Cholesterol-loaded Cyclodextrin Research Articles

Novel technical strategies to optimize cryopreservation of goat semen using cholesterol-loaded cyclodextrin

Artificial insemination is well-established in dairy cattle, with sires housed in commercial studs for processing. In some species, however, sires located on-farm are used for artificial insemination by shipping their semen to an off-site laboratory for processing within 24 h of collection. To expedite semen transport from the farm to laboratory, protocols must be uncomplicated. For goat semen, an obstacle is the seminal plasma, which must be removed because it contains proteins that impede sperm quality. Our objective is to develop a simple strategy to transiently store goat semen for 24 h prior to freezing. Cholesterol-loaded cyclodextrin (CLC) has been demonstrated to improve sperm tolerance to cryopreservation. Therefore, we hypothesized that CLC improves goat sperm resistance to seminal plasma damage, over 24 h prior to cryopreservation. We first evaluated the ability of CLC to protect goat sperm against seminal plasma damage by treating fresh semen with or without seminal plasma prior to cryopreservation. Second, fresh goat semen with seminal plasma was extended in skim milk-based extender ± CLC and held for 24 h at 5 °C prior to freezing. Our results indicate that CLC treatment improves goat sperm resistance to seminal plasma-mediated injury and protects sperm quality over 24 h prior to freezing (P < 0.05). Although the in vivo fertility of semen must first be assessed, it is possible that protocols for goat semen cryopreservation can be simplified by including CLC and eliminating seminal plasma removal. Processing and distribution of goat semen for AI would thereby be facilitated.

The effect of cholesterol loaded cyclodextrins on post-thawing quality of buffalo semen in relation to sperm DNA damage and ultrastructure

The cryopreservation of germ cells is a major tool for the propagation of animals with desired genetic traits. Although cryopreservation of spermatozoa in some animals is effective, its effectiveness is variable. For example, cryopreservation efficiency of buffalo bull spermatozoa remains very poor. In this study, we evaluated sperm DNA damage and ultrastructure in buffalo bull spermatozoa vitrified in the presence or absence of cholesterol-loaded cyclodextrins (CLC). Our results showed that cryopreserved buffalo spermatozoa had elevated levels of deteriorated plasma and mitochondrial membranes, which are the likely causes of DNA damage after vitrification. Accordingly, the levels of the activity of Alanine Aminotransferase (ALT), Alkaline phosphatase (ALP) and Aspartate Aminotransferase (AST) were also elevated following exposure of buffalo bull spermatozoa to a cycle of freezing-thawing. Importantly, supplementation of Tris-Egg Yolk-Glucose (TEYG) extender with (CLC) improved the quality of buffalo spermatozoa following cryopreservation. This protective effect of CLC is likely due to decreasing mitochondrial and plasma membrane deterioration with subsequent inhibition of DNA damage. These results suggest that cholesterol loss is the likely reason for poor semen quality in buffaloes following cryopreservation, and provide evidence that manipulating lipid content during cryopreservation is a promising strategy to improve the quality of buffalo semen.

Cholesterol-loaded cyclodextrin improves ram sperm cryoresistance in skim milk-extender

Cholesterol-loaded cyclodextrin (CLC) is known to improve ram sperm cryosurvival. This study expands on previous research to: (1) determine the mechanism by which CLC improves ram sperm cryosurvival and (2) compare the efficiency of a novel, skim milk-based extender containing CLC to a traditional egg yolk-based extender. Hypothesis #1 was that CLC enhances membrane cholesterol content to increase the resistance of ram sperm to cold and osmotic stress, thereby improving cryosurvival. We first assessed the ability of fresh sperm treated with CLC to withstand cold shock. Second, fresh sperm were treated with CLC to evaluate their tolerance to osmotic stress. Third, to confirm that cholesterol is incorporated into the sperm using CLC, we quantified sperm cholesterol. To test Hypothesis #2 that CLC is most effective in a medium without competing cholesterol, we compared sperm cryosurvival and fertility in skim milk-based extender containing CLC versus in a traditional egg yolk-based freezing extender without CLC. Our data confirmed that CLC treatment improves ram sperm cold shock and osmotic stress resistance, and augments sperm cholesterol content. Semen in skim milk-based extender containing CLC prior to freezing, had more motile sperm with intact acrosomes after thawing compared to semen in egg yolk-based extender. In contrast, sperm plasma membrane integrity and in vivo fertility of the semen cryopreserved in the skim milk-based extender with CLC did not differ from semen that was cryopreserved in egg yolk-based extender. Further research is warranted to combine CLC with other cryoprotection strategies or to modify the insemination protocol.

Effect of cholesterol loaded cyclodextrin (CLC) on lipid peroxidation and reactive oxygen species levels during cryopreservation of buffalo (Bubalus bubalis) spermatozoa

ObjectiveTo assess the effect of cholesterol loaded cyclodextrin on seminal attributes (progressive motility, livability, hypo-osmotic swelling (HOS) response and acrosomal integrity), lipid peroxidation (LPO) and reactive oxygen species (ROS) levels during cryopreservation of buffalo (Bubalus bubalis) spermatozoa. MethodsA total of 24 ejaculates (mass motility ≥4; progressive motility ≥80%) were collected from three murrah buffalo bulls (8 from each bull) through artificial vagina twice a week. One part of semen was diluted with TEYC extender (group I) and other part was treated with cholesterol loaded cyclodextrin before final dilution with TEYC extender (group II). Semen samples were evaluated for various seminal attributes, LPO and ROS levels at fresh, pre-freeze and post-thaw stage in group I and group II. ResultsSeminal attributes (progressive motility, livability, hypo-osmotic swelling (HOS) response and acrosomal integrity) were significantly higher in group II than group I at pre-freeze (P < 0.05) and post-thaw (P < 0.01) stage. LPO and ROS levels were significantly higher in group I as compared to group II at pre-freeze (P < 0.05) and post-thaw (P < 0.01) stage. ConclusionIt is concluded that cholesterol loaded cyclodextrin reduces LPO and ROS levels during cryopreservation of buffalo spermatozoa.

Cholesterol-loaded cyclodextrins prevent cryocapacitation damages in buffalo (Bubalus bubalis) cryopreserved sperm

The aim of this study was to investigate the effect of cholesterol-loaded cyclodextrins (CLC) on motility, viability, capacitation status, and in vivo fertility of buffalo frozen-thawed sperm. After the initial semen assessment, buffalo sperm were diluted in BULLXcell extender containing 0- (control), 1.5-, and 3-mg/mL CLC and cryopreserved. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability-capacitation status was assessed by Hoechst 33258-chlortetracycline (CTC) assay. Capacitation status was also evaluated by an indirect immunofluorescence assay to localize phosphotyrosine-containing proteins. Moreover, buffaloes were artificial inseminated to assess the in vivo–fertilizing potential of CLC-treated semen. No differences among control, 1.5-, and 3-mg/mL CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). However, the extender supplementation with CLC significantly reduced sperm cryocapacitation. Indeed, CLC treatment decreased (P < 0.01) the proportion of sperm showing the CTC pattern B (capacitated sperm) compared with the control (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7, respectively, with 0, 1.5-, and 3-mg/mL CLC; P < 0.01). Furthermore, the percentage of sperm displaying tyrosine-phosphorylated pattern EA (i.e. high capacitation level) was reduced (P < 0.01) in both CLC-treated groups (10.8 ± 3.3 and 5.6 ± 1.6, respectively, with 1.5- and 3-mg/mL CLC) compared with the control (37.3 ± 6.9), reaching values similar to those recorded in fresh semen (11.0 ± 3.5). In addition, treating sperm with 3-mg/mL CLC increased (P < 0.01) the percentage of nonfluorescent (pattern NF), i.e., non-capacitated sperm (41.8 ± 3.6) compared with fresh semen (11.0 ± 6.9). No differences were recorded in pregnancy rates at 60 days post-artificial insemination among control, 1.5- and 3-mg/mL CLC groups (59.7%, 65.6%, and 56.9%, respectively). In conclusion, CLC treatment of buffalo sperm strongly decreases sperm cryocapacitation damages, without affecting the in vivo fertilizing capability.

Pretreatment of Addra gazelle (Nanger dama ruficollis) spermatozoa with cholesterol-loaded cyclodextrins improves cryosurvival

Preserving genetic diversity of the critically endangered Addra gazelle (Nanger dama ruficollis) could be enhanced through the use of frozen-thawed sperm and artificial insemination. Our aim was to characterize Addra ejaculate traits and to assess the effects of cholesterol-loaded cyclodextrin (CLC) on sperm cryosurvival. Fresh ejaculates were treated with CLC (0, 0.5, 1.5, 3.0, and 6.0 mg/ml) prior to cryopreservation. All males produced spermic ejaculates with >75% sperm motility. The mean ± SEM seminal volume, sperm concentration, percent motility, forward progression, and percent morphologically normal spermatozoa were 3.2 ± 0.3 ml, 1.2 ± 0.3 × 109, 75.82 ± 2.7%, 3.2 ± 0.3 (0–5 scale; 5 = most progressive), and 57.12 ± 3.8%, respectively. More than 92% contained an intact acrosome. There was no effect of time or in vitro incubation on progression or acrosomal integrity on thawed samples (P > 0.05). Spermatozoa pre-treated with 0.5 mg/ml CLC retained higher (P < 0.05) motility post-thaw than aliquots treated with 0, 3.0, or 6.0 mg/ml of CLC. Spermatozoa pre-treated with 0.5, 1.5, or 3.0 mg/ml CLC exhibited greater viability than counterparts (P < 0.05). Sperm kinetics including beat cross frequency (BCF), average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL) did not differ among samples (P > 0.05). Linearity (LIN) and straightness (STR) were different among samples after thawing. Results demonstrate treatment with CLC (0.5 mg/ml) protects Addra spermatozoa from cryo-damage. Reported advances will facilitate establishment of a frozen repository and support the genetic management of this critically endangered north African desert antelope.

Evaluation of cholesterol- treated dromedary camel sperm function by heterologous IVF and AI

Cholesterol (cholesterol-loaded cyclodextrins: CLC) treatment of dromedary camel sperm prior to freezing enhances cryosurvival. The present study first validated the efficacy of a heterologous zona-free goat oocyte assay (n=115 oocytes) to evaluate camel sperm function in vitro (Experiment 1: n=6 bulls), then examined the effects of CLC treatment (1.5mg/mL CLC; CLC+) versus no treatment (0 CLC) of fresh (Experiment 2: n=4 bulls) and frozen-thawed (Experiment 3: n=5 bulls) camel sperm to penetrate, de-condense and form pro-nuclei in in vitro-matured goat oocytes. Finally, the ability of fresh 0 CLC and CLC+ sperm to fertilize in vivo was studied by artificially inseminating super-ovulated females (n=7–9 per treatment) and examining embryo production (Experiment 4: n=4–5 bulls/treatment). Camel spermatozoa penetrated (60%) and formed pro-nuclei (33%) in goat oocytes demonstrating the utility of this heterologous system for assessing sperm function in vitro. For fresh spermatozoa, 0 CLC-treated sperm performed better than their CLC+ counterparts for all parameters measured (P<0.05). In contrast, cryopreservation resulted in a sharp decline in sperm-oocyte interaction in 0 CLC aliquots but remained unaltered in CLC+ aliquots demonstrating a protective effect of cholesterol treatment. There was no difference between treatments in the in vitro fertilizing ability of frozen-thawed sperm or in the numbers of embryos retrieved following AI with fresh 0 CLC or CLC+ sperm. We conclude that although CLC treatment of dromedary camel sperm improves sperm motility it fails to confer an advantage to them in terms of improved in vitro sperm-oocyte interaction or in vivo fertilization under the conditions tested.

Survival of buffalo bull spermatozoa: effect on structure and function due to alpha-lipoic acid and cholesterol-loaded cyclodextrin

Sperm survival depending upon integral membranes and function is imperative for fertilization. This study was designed to augment survival of buffalo spermatozoa using alpha-lipoic acid (ALA) and cholesterol-loaded cyclodextrin (CLC) during cryopreservation. Semen was frozen using 0, 0.5, 1, 1.5, 2 and 2.5mmolL-1 ALA (experiment 1) and ALA or CLC separately or together (experiment 2). Semen was assessed for post-thaw motility, plasma membrane integrity (PMI), intact acrosome and plasma membrane (IACR-IPM) and DNA integrity at 0, 1.5, 3 and 4.5hr of incubation. In experiment 1, use of 0.5mmolL-1 ALA enhanced the sperm cryosurvival and post-thaw longevity than other groups up to 4.5hr of incubation, and this concentration of ALA was used in second experiment with CLC. The results revealed higher (p<.05) sperm survival function and time of sperm attributes due to use of ALA than CLC and control. However, the sperm quality did not improve (p>.05) when ALA was combined with CLC. In conclusion, survival of buffalo bull spermatozoa during freeze-thawing and post-thaw incubation can be enhanced more with ALA than CLC or control, followed by CLC than control. However, there is no synergistic effect on survival of buffalo bull spermatozoa due to ALA and CLC.

Effect of cyclodextrins, cholesterol and vitamin E and their complexation on cryopreserved epididymal ram semen

The aim of the present study was to investigate the potential benefit of vitamin E and cholesterol when preloaded in cyclodextrins, alone or in association to protect ram epididymal sperm during the freezing-thawing process. Epididymal sperm were collected from Twenty four testes; sperm from the two testicles of each ram was pooled and divided in 7 aliquots. The control aliquot was diluted with Fraction A (Tris-citric acid-fructose) without further supplementation. The Six (6) other aliquots were diluted with fractions A containing cyclodextrins (CD), cholesterol (CHL), vitamin E (Vit E), cholesterol-loaded cyclodextrins (CD-CHL), vitamin E-loaded cyclodextrins (CD-Vit E) and CD-CHL and CD-Vit E (CD-CHL-Vit E), respectively. After incubation at 22°C for 15min and addition of Fraction B (Fraction A-egg yolk-glycerol), all aliquots were equilibrated at 4°C for 2h and then frozen in liquid nitrogen. A Computer Aided Semen Analysis (CASA) was used to investigate the impact on different motility parameters and the hypo-osmotic swelling test (HOST) to quantify membrane functionality. The Oxidative stress impact on sperm membrane was investigated through lipid peroxidation (LPO) measurement. After thawing, CD-Vit E and CD-CHL treatments improved significantly (P<0.05) the total motility, VAP and linearity (LIN), compared to the control, Vit E and CHL samples. However, the association of CD-CHL and CD-Vit E (CD-CHL-Vit E) exhibited a significant effect on total motility, progressive motility, membrane functionality, sperm velocities (VCL, VSL and VAP) and LIN (P<0.05). Membrane lipid peroxidation was significantly lower in semen pretreated with CD-Vit E than in control and Vit E alone. Among all treatments, the association of CD-CHL and CD-Vit E (CD-CHL-Vit E) showed the highest protection against LPO (P<0.05). The present results revealed that the significant impact was observed when vitamin E, cholesterol and cyclodextrins are all used in the same treatment, thus demonstrating the complementary effect of solubilized vitamin E and cholesterol in protecting concomitantly spermatozoa against cold shock and oxidative stress.

Modification of membrane cholesterol and its impact on frozen-thawed chicken sperm characteristics.

This study was conducted to determine the changes in chicken sperm plasma membranes fluidity and polarity as lipid packing arrangement induced by cholesterol-loaded cyclodextrin (CLC) and 2-hydroxypropyl-β-cyclodextrin (HBCD) and how sperm cryopreservation outcomes are improved by these changes. Treatment with 2 mg HBCD supported the highest (P < 0.01) percentage of viable spermatozoa compared with the control and CLCs groups after cryopreservation. The percentage of post-thaw progressive and rapid sperm motility was highest in 2 mg HBCD (P < 0.01). After thawing, sperm treated with 1 or 2 mg CLC showed the highest anisotropy at 5, 21, 25 and 40°C (P < 0.01). At 25°C, the lowest anisotropy was observed in the thawed semen from the control group. The highest value (P < 0.01) of generalized polarization (GP) (0.5) at 5°C was observed in the 1 mg CLC treated sample. After 2 h of incubation, the highest percentage of viable spermatozoa was observed in the HBCD group in relation to the other treatments (P < 0.01). Exposure to 1 mg or 2 mg of CLC significantly decreased the percentage of live spermatozoa after thawing (P < 0.01). In conclusion, HBCD appears to play a role in the modification of sperm membranes, increasing their fluidity and preventing them against membrane phase transition to gel, thus minimizing freezing-thaw sperm damage. HBCD treatment enhances chicken sperm viability and motility after cryopreservation and subsequent storage. This novel procedure may be useful for improving the technology for cryopreservation of fowl spermatozoa.

Cholesterol-Loaded Cyclodextrin Increases the Cholesterol Content of Goat Sperm to Improve Cold and Osmotic Resistance and Maintain Sperm Function after Cryopreservation.

The success of semen cryopreservation depends on sperm membrane integrity and function after thawing. Cholesterol-loaded cyclodextrin (CLC) is used for in vitro incorporation of cholesterol to protect cells against cold temperatures. We hypothesized that CLC treatment also enhances sperm cholesterol content to increase tolerance to osmotic shock and cryoresistance, thereby improving fertility. We confirmed the fact that treatment of goat semen with 3 mg/ml CLC increases sperm cholesterol content using both the Liebermann-Burchard approach and filipin III labeling of membrane cholesterol. Sperm were then treated with or without CLC and cryopreserved. After thawing, sperm cholesterol dramatically fell, even in the presence of CLC, which explains the mechanism of cryocapacitation. CLC treatment, however, maintained a normal prefreeze cholesterol level in sperm after cryopreservation. Furthermore, fresh sperm treated with CLC and subjected to either cold shock or incubated in hypo-, iso-, and hyperosmotic media, designed to mimic stresses associated with freezing/thawing, displayed increased temperature and osmotic tolerance. CLC treatment also improved sperm viability, motility, and acrosome integrity after thawing. Furthermore, CLC treatment did not affect the sperm's ability to undergo in vitro capacitation according to chlortetracycline fluorescence and protein tyrosine phosphorylation. A pilot field trial demonstrated that artificial insemination with sperm that underwent increased cholesterol levels following CLC treatment yielded higher fertility ( ITALIC! P< 0.1) and proliferation ( ITALIC! P< 0.05) rates in vivo than untreated semen from the same ejaculate samples. These observations suggest that CLC treatment could be used to improve cryoprotection during the freezing and thawing of goat sperm.

Effect of incubation on freezability of cholesterol-loaded cyclodextrin treated buffalo (Bubalus bubalis) spermatozoa.

Aim:The aim of this study was to investigate the effect of incubation on freezability of cholesterol loaded cyclodextrin (CLC) treated buffalo spermatozoa.Materials and Methods:Semen samples with mass motility of 3+ and greater, collected from Murrah buffalo bulls were utilized. Immediately after collection, four equal groups of semen sample were made. Group I was kept as control and diluted with Tris upto concentration of 60×106 sperm/ml, where as Groups II, III, and IV were treated with CLC at 3 mg/120× 106 spermatozoa, incubated at 37°C for action of CLC for 10, 15 and 20 min, respectively, and diluted with tris upto concentration of 60×106 sperm/ml. All groups were subjected to equilibration and freezing. The evaluation of semen samples from all groups was carried out at fresh, pre-freeze and post-thaw stage for progressive motility, viability and hypo-osmotic swelling response (HOS response).Results:At the pre-freeze stage, significantly (p<0.05) higher percentage of progressive motility and viability was observed in treatment groups as compared to control with no significant difference among treatment groups. HOS response was significantly (p<0.05) higher in treatment groups as compared to control at pre-freeze stage. At post-thaw stage, significantly (p<0.05) higher percentage of progressive motility, viability and HOS response was recorded in Group II as compared to control and other treatment groups (III and IV). Group II retained significant post-thaw motility and viability at various post-thaw incubation periods.Conclusion:Incubation period of 10 min for CLC treated buffalo spermatozoa yielded significantly higher results in terms of freezability as compared to incubation for 15 and 20 min.

Effect of incubation on freezability of cholesterol-loaded cyclodextrin treated buffalo (Bubalus bubalis) spermatozoa

Effect of incubation on freezability of cholesterol-loaded cyclodextrin treated buffalo (Bubalus bubalis) spermatozoa

Effect of different sugars supplemented to the extender in combination with cholesterol-loaded cyclodextrin (CLC) on post-thaw quality of ram spermatozoa

The present study was designed to determine the effect of different sugars supplemented to the extender on post-thaw quality of CLC treated ram sperm. The ejaculates (n=10) were divided into two aliquots: first aliquot was treated with CLC (3mg/120×106 sperm), whereas, second aliquot remained untreated and served as control. The samples were then incubated at 35°C for 15min. After incubation, each aliquot was again divided into 3 equal subgroups. Each of the subgroups was then diluted with one of the extenders containing fructose, sucrose or trehalose (28mM) to make a final sperm concentration 200×106/ml. After dilution, the semen samples were loaded into 0.25ml straws and cryopreserved using standard procedure. The data showed that type of the sugar did not affect post-thaw sperm parameters (P>0.05). However, the CLC pre-treatment improved (P<0.05) motile, live, live-intact, total intact and the number of total active sperm per straw. A non-significant interaction (P>0.05) was determined between type of sugar and CLC. In conclusion, CLC pre-treatment significantly improved post-thaw sperm parameters in ram and has no interaction with any of the used sugars.

Cholesterol-loaded cyclodextrin in fresh goat sperm improves cryosurvival rates

This study evaluated the effect of adding different concentration of cholesterol-loaded-cyclodextrin (CLC) on sperm quality after thawing. Thirty ejaculates were diluted, centrifuged and resuspended to 120 million cells/mL in a Tris diluent. Semen was treated with 0 (Control), 0.75, 1.5, 3.0, 4.5, 6.0 or 7.5 mg of CLC. Then, the samples were cooled at 4 °C for 2 h, diluted with Tris-egg yolk and 2% glycerol, packaged into 0.5 mL straws, frozen in liquid nitrogen (N2 ) vapor for 20 min before being plunged into N2 . Straws were thawed at 37 °C for 30 sec, and evaluated for thermal resistance test (TRT); progressive motility using CASA; hypoosmotic test and for binding capacity of sperm to perivitelline membrane (PM). The variables were determined using ANOVA at 5% probability. Higher percentage of motile sperm were maintained after thawing and TTR when 0.75 mg CLC was added, evaluated at 0, 60 and 120 min of incubation (50.4, 33.8 and 22.5%, respectively) compared to other treatments (P<0.05). The percentage of coiling was higher in sperm treated with 6.0 and 7.0 mg of CLC than other treatments (P<0.05). Addition of 0.75 mg CLCs also resulted in more sperm binding to the PM after cryopreservation than control sperm (166 vs 65; P<0.05). However, when the spermatozoa binding potential was determined on a motile sperm basis by dividing the average number of spermatozoa bound to PM for each bucks by the percentage of motile spermatozoa, CLC treatment provided higher binding efficiency (1.52) than control (1.00; P<0.05). In conclusion, CLCs improved the percentage of post-thaw of motility in caprine sperm as well as increased the number of sperm that bind to PM. Addition of 0.75 mg of CLC to caprine sperm prior to cryopreservation improved the quality sperm motility for up to 2 h.

40 CHOLESTEROL SUPPLEMENTATION REDUCES CRYOCAPACITATION DAMAGES IN BUFFALO (BUBALUS BUBALIS) SPERM

Buffalo sperm are more sensitive to cryopreservation and thawing-induced damages than cattle sperm, thus resulting in lower fertilizing ability. Cryopreservation induces considerable capacitation-like changes in buffalo sperm (Elkhawagah et al. 2014 J. Buffalo Sci. 3, 3–11). It is known that specific lipid mixtures, including cholesterol, stabilise and protect spermatozoa from freeze-thaw damage (Rajoriya et al. 2014 Vet. World. 7, 702–706). Therefore, the aim of this study was to investigate the effect of cholesterol loaded cyclodextrins (CLC) before cryopreservation on the capacitation status of buffalo frozen sperm. Four ejaculates from 4 bulls were selected, split in 3 aliquots, and diluted at 37°C with BULLXcell extender, containing 0 (control), 1.5, and 3 mg mL–1 CLC, to a final concentration of 30 × 106 sperm mL–1. Cyclodextrin was loaded with cholesterol as described earlier (Purdy and Graham 2004 Cryobiology 48(1), 36–45). The aliquots were frozen according to standard procedures. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability and capacitation status were evaluated by Hoechst 33258/CTC. Briefly, Percoll separated spermatozoa were incubated with 1% (wt/vol) Hoechst 33258 in a protein-free medium. After centrifugation (900 × g, 5 min), the pellet was resuspended in CTC staining solution (750 mM CTC, 5 mM cysteine in 130 mM NaCl, and 20 mM Tris acid, pH 7.8), fixed with glutaraldehyde (12.5% vol/vol), and placed on a slide. At least 100 sperm per slide were analysed and classified into 1 of 3 CTC staining patterns: pattern F, with fluorescence over the entire sperm head (noncapacitated); pattern B, with a fluorescence-free band in the postacrosomal region (capacitated); pattern AR, with no acrosome and a thin band of fluorescence along the equatorial segment (acrosome reacted). Data were analysed by ANOVA. No differences among control, 1.5, and 3 mg mL–1 CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). The most interesting result arising was the strong reduction of sperm cryocapacitation observed when the extender was supplemented with CLC, as shown by the increased percentage of sperm displaying pattern F (28.6 ± 3.3, 61.5 ± 1.7, and 47.6 ± 4.3 in control, 1.5, and 3 mg mL–1 CLC groups, respectively; P &lt; 0.01) and the decreased percentage of sperm displaying pattern B (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7 in control, 1.5, and 3 mg mL–1 CLC groups, respectively; P &lt; 0.01) compared with the control. The maximum beneficial effect on semen cryopreservation was recorded with the dose of 1.5 mg mL–1 CLC. However, no differences were detected in pattern AR that remained low in all groups (1.8 ± 0.7, 0.8 ± 0.4, and 1.1 ± 0.6 in the control, 1.5, and 3 mg mL–1 CLC groups, respectively). In conclusion, it was demonstrated that treating buffalo sperm with cholesterol before cryopreservation strongly decreases cryocapacitation damages, likely by stabilising sperm membrane, hence improving sperm quality.

39 FERTILITY POTENTIAL OF FROZEN-THAWED WOOD BISON SEMEN USING EXTENDER WITHOUT EXOGENOUS PROTEIN

Cryopreservation of semen free of infectious agents is critical for the recovery and preservation of genetic diversity in Canada’s threatened wood bison populations. Egg yolk is a common constituent of conventional semen extenders, but it raises biosafety concerns related to transmission of infectious agents. The purpose of this experiment was to determine the fertility potential, both in vitro and in vivo, of semen frozen without the use of exogenous protein but with addition of cholesterol loaded cyclodextrin (CLC). Semen was collected by electro ejaculation from 4 wood bison bulls. Fresh sperm total motility and concentration were analysed using a computer-assisted sperm analyzer. Ejaculates with total sperm motility of &gt;60% and a concentration of &gt;200 × 106 sperm mL–1 were selected and pooled among bulls. For the control group, pooled semen was diluted to 50 × 106 sperm mL–1 with conventional extender containing 20% egg yolk and 7% glycerol. For the test group, pooled semen was diluted to 100 × 106 sperm mL–1 in Tris-citric acid buffer and incubated with 2 mg mL–1 CLC for 15 min before 1 : 1 dilution with 14% glycerol extender. Extended semen was placed in 0.5-mL straws, cooled, and frozen. Post-thaw motility analysis was conducted using a computer-assisted sperm analyzer, and the 2 collections with highest post-thaw motility were selected for fertility testing. Heterologous IVF was conducted using bovine oocytes obtained from an abattoir. After 20 to 22 h of in vitro maturation, the cumulus-oocyte complexes were placed into drops with either control or test semen. After 18 h, potential zygotes were denuded and moved to culture media. Cleavage and blastocyst rates were assessed on Day 4 and 8, respectively, from oocyte collection. The fertility potential of the semen was also tested in vivo using synchronized wood bison cows. At 24 h after hCG treatment, bison cows were assigned randomly to 2 groups and inseminated twice, 12 h apart, with frozen-thawed control semen (n = 23) or test semen (n = 23). Pregnancy was assessed 34 to 36 days after insemination by transrectal ultrasonography. Total sperm motility (mean ± standard error of the mean) of fresh semen was 80.2 ± 3.3%. The post-thaw motility was 41.7 ± 2.9% and 44.6 ± 3.3% for control and test semen, respectively. The cleavage rates of bovine oocytes fertilized in vitro with bison semen from the control group (n = 278) and test group (n = 299) were 49.9 ± 2.1% and 41.3 ± 4.5%, respectively, and the blastocyst rates were 17.7 ± 1.7% and 17.3 ± 2.5%, respectively. Pregnancy rates of wood bison artificially inseminated with control and test semen were 39 and 0%, respectively. In conclusion, wood bison semen frozen with CLC (without exogenous protein) resulted in post-thaw motility and in vitro fertility potential comparable to that of conventional egg yolk extender. However, CLC semen failed to fertilize in vivo, perhaps because of disruption of the processes of capacitation. The dichotomy between in vivo and in vitro results was surprising. It may be necessary to exercise caution when using IVF as a tool to assess fertility in vivo.

122 TIMED ARTIFICIAL INSEMINATION IN WOOD BISON USING FROZEN-THAWED SEMEN

Recent progress with methods to control ovulation and semen cryopreservation in Wood Bison was the impetus to test the feasibility of timed AI to facilitate reclamation of this threatened species. A 2 × 2 design was used to compare the efficacy of 2 ovulation synchronization techniques and 2 semen cryopreservation protocols. Female Wood Bison were assigned randomly to 2 groups (n = 24/group) in which ovarian synchronization was induced by ultrasound-guided ablation of follicles &gt;5 mm or intramuscular treatment with 2.5 mg of estradiol 17B + 50 mg of progesterone (E+P) in canola oil. A progesterone-releasing intravaginal device (PRID) was placed at the time of follicle ablation (for 5 days) or E+P treatment (for 8 days) in the respective groups. A luteolytic dose of prostaglandin was given at the time of PRID removal, and 2500 IU of hCG was given IM 3 days later. Bison were inseminated 24 and 36 h after hCG treatment using frozen-thawed semen. The semen was collected by electro-ejaculation from 4 Wood Bison bulls, pooled, and divided into aliquots diluted in either egg-yolk extender (EY) or cholesterol-loaded cyclodextrin extender (CLC). Half the bison in each synchronization group were inseminated with either EY- or CLC-extended semen. Bison were examined by ultrasonography every 12 h beginning on the day of hCG treatment for 3 days or until ovulation was detected, whichever occurred first. Pregnancy diagnosis was made by ultrasonography 34–36 days after insemination. Two bison were excluded during the experiment because of handling difficulty; therefore, the total number of bison used was 46. Ovulation rate and interval to ovulation were compared between synchronization groups by chi-square and t-test, respectively. Pregnancy rates were compared among groups by 2-way ANOVA after transforming data to arcsin. The ovulation rate was not different between synchronization groups [combined mean, 37/46 (80%)], nor was the degree of synchrony, as assessed by the residuals (variation from the mean) in the respective groups. However, the diameter (mean ± standard error of the mean) of the dominant follicle at the time of hCG treatment was smaller in the follicle ablation group than in the E+P group (10.5 ± 0.6 v. 13.9 ± 0.6; P &lt; 0.04), and the interval from hCG treatment to ovulation tended to be longer (35.3 ± 1.6 v. 31.8 ± 1.3 h; P ≤ 0.10). Pregnancy rate was not affected by synchronization procedure, but pregnancy was detected only in the EY-inseminated group (9/23 v. 0/23; P &lt; 0.01). Despite that post-thaw sperm motility was similar for EY and CLC semen (41.7 ± 2.9 and 44.6 ± 3.3%; respectively), CLC-treated semen failed to impregnate bison in vivo. We concluded that synchronization and timed insemination with frozen-thawed semen is feasible in Wood Bison. Of the 23 bison inseminated with EY-extended semen, 21 ovulated (91%), and of those that ovulated 9 became pregnant (43%). Both synchronization schemes were effective, but the ablation protocol may be improved by an additional day between ablation and hCG treatment. We thank Vetoquinol Canada and Merck Animal Health for providing hormone treatments.

Effect of cholesterol supplementation on cryosurvival of goat spermatozoa.

Aim:Sperm membrane cholesterol influences cryodamage during cryopreservation. The present study was carried out to evaluate the effect of varying cholesterol levels in Tris based extenders on the freezability of sexually healthy Malabari buck semen.Materials and Methods:A total of 48 ejaculates from two adults healthy sexually healthy Malabari bucks were utilized for the study. The collected and pooled ejaculates were divided into four groups with Group I serving as Control - I, Group II and III were treated with 1 mg and 2 mg of cholesterol-loaded-cyclodextrin (CLC)/120 × 106 spermatozoa, respectively, and Group IV, treated with 1 mg methyl-β-cyclodextrin (MβCD) served as Control - II. Manual freezing was carried out to cryopreserve the treated and control spermatozoa.Results:Treatment of semen samples with CLC resulted in improved maintenance of sperm motility at pre-freeze and post-thaw stages of cryopreservation without affecting hypo-osmotic swelling response. Treatment of semen with 1 mg of CLC/120 × 106 spermatozoa was observed to be better than treatment with 2 mg of CLC/120 × 106 spermatozoa. In general, MβCD treatment was found to result in significantly lower sperm characteristics than those of Control - I and CLC treatment at pre-feeze and post-thaw stages and when incubated up to 4 h.Conclusion:Cholesterol treatment of sexually healthy Malabari buck semen was found to hold promise for improving cryopreservability of spermatozoa.

Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa

Buffalo spermatozoa are comparatively more susceptible to freezing hazards than cattle spermatozoa. In recent times incubation of spermatozoa with cholesterol-loaded-cyclodextrins (CLC) has shown improvements in semen quality in several species. Therefore, this study was undertaken to evaluate the incubation level of CLC at which maximum benefit is derived for the buffalo spermatozoa. For the study, 120 million spermatozoa were incubated in 2, 3 and 4mg/mL of CLC (Gr II, III and IV, respectively) and cholesterol and phospholipids content, their ratio, flow cytometric evaluation of plasma membrane integrity (PMI), plasma membrane fluidity and extent of cryoinjury (Chlortetracycline, CTC assay) were compared with an untreated control (Gr I). Additionally the ability of cholesterol-loaded-spermatozoa to undergo induced acrosome reaction (IAR) using ionophore calcium (A23187) was evaluated in frozen–thaw samples. Data show a significant and linear increase (CV=0.88) in cholesterol content of spermatozoa in Gr II, III and IV and a significant decrease in phospholipids content at frozen–thaw stage in Gr IV than Gr III spermatozoa. The study revealed a significant improvement in PMI and significant reduction in plasma membrane fluidity and cryoinjury of CLC treated spermatozoa at progressive stages in three groups compared to control. Nevertheless, spermatozoa of Gr II, III and IV were significantly less responsive to ionophore calcium (A23187) than Gr I. This study shows for the first time that incubation of buffalo bull spermatozoa with CLC (3mg/120×106) prior to processing permits greater numbers of sperm to survive cryopreservation while allowing spermatozoa to capacitate and the acrosome to react to AR inducer ionophore calcium (A23187).