Egg yolk plasma can replace egg yolk in stallion freezing extenders

Hen egg yolk is normally used as a cryoprotective agent in semen freezing extenders, but its use has sanitary and practical disadvantages. Moreover the protection afforded by egg yolk has not yet been completely elucidated. The objective of this study was to compare the egg yolk plasma fraction to whole egg yolk in stallion freezing extender. Plasma contains mainly Low Density Lipoproteins (LDL), which are widely presumed to be the cryoprotective agent in egg yolk. Plasma can be produced on an industrial scale, sterilised by gamma-irradiation and incorporated in a ready-to-use extender (our ultimate objective). Plasma samples were subjected to different doses of gamma-irradiation (3, 5, 10 kGy) without dramatic chemical changes that may affect their cryoprotective properties. Stallion semen was frozen with whole egg yolk as a control and with sterilised egg yolk plasma. A fertility trial was conducted on a total of 70 mares' cycles. Fertility per cycle was 60% after insemination of semen frozen in our control extender containing egg yolk (EY), compared to 69% for the extender containing sterilised egg yolk plasma (EYP) (P > 0.05). Post-thaw motility and membrane integrity of spermatozoa were also analysed. Motility parameters were not significantly different between extenders except for the variable VAP (for EY versus EYP, VAP: 63 μm.s⁻¹ versus 59 μm.s⁻¹, a, b: P < 0.001; PROG: 41% versus 39%, RAP30: 56% versus 54%; RAP40: 51% versus 48%, P > 0.05). Membrane integrity was better preserved in EY than in EYP but the difference between extenders was small (P < 0.05). Our results demonstrated that sterilised egg yolk plasma has the potential to replace egg yolk in stallion freezing extender. This experiment led to the development of a ready-to-use extender called INRA-Freeze^® (IMV-Technologies, France).     

Evaluation of duck egg yolk for the cryopreservation of Nili-Ravi buffalo bull semen

This study was carried out to investigate if the substitution of chicken egg yolk (CEY) with duck egg yolk (DEY) in extenders can improve the quality of frozen-thawed semen of Nili-Ravi buffalo bulls and to study if reducing DEY level in extender affects the freezability results. Thirty semen samples collected from three buffalo bulls were diluted in extenders A, B, C, D and E containing tris, citric acid, fructose, egg yolk, glycerol and antibiotics. Extender A contained 20% CEY (control), while extenders B, C, D and E contained 5, 10, 15 and 20% DEY, respectively. After freezing and storage for 24h in liquid nitrogen, samples were evaluated for post-thaw quality. The post extension sperm motility did not differ between extenders A (control) and E (20% DEY). The same was true for post-thaw percentage of sperm with functional plasma membrane and percentage of sperm with abnormal heads or mid pieces. However, extender E showed higher (P<0.05) values for post-thaw sperm motility, livability and absolute index of livability of spermatozoa at 37 °C compared to extender A. Spermatozoa with abnormal tail were lower (P<0.05) in extender E compared to extender A. Values of these parameters of post-thaw semen quality were highest for extender E containing 20% DEY and decreased significantly with decrease in the concentration of DEY, except sperm abnormalities (head, mid-piece and tail) which increased with decrease in DEY level. These results showed that replacement of 20% CEY with 20% DEY in extenders significantly improved post-thaw sperm motility, livability and absolute index of livability of spermatozoa and reduced tail abnormalities. Reduction in the level of DEY in extenders from 20% adversely affected post-thaw semen quality of Nili-Ravi buffalo bulls.     

Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa

Four experiments were conducted to investigate the effects of egg yolk during the freezing step of cryopreservation (namely, the process except for the cooling step), on the viability of goat spermatozoa. The effects of egg yolk on sperm motility and acrosome integrity during the freezing step were investigated in Experiment 1. Spermatozoa diluted with Tris-citric acid-glucose (TCG) solution containing 20% (v/v) egg yolk were cooled to 5 degrees C, washed, and then frozen in TCG with egg yolk (TCG-Y), TCG without egg yolk (TGG-NY), 0.370 M trehalose with egg yolk (TH-Y), or trehalose without egg yolk (TH-NY). All extenders contained glycerol. In frozen-thawed spermatozoa, the inclusion of egg yolk in the freezing extenders increased (P<0.05) percentages of motile sperm, progressively motile sperm, and the recovery rate (ratio of post-thaw to pre-freeze values), but decreased (P<0.05) acrosomal integrity. Moreover, extenders with trehalose had better (P<0.05) post-thaw sperm viability. In Experiment 2, the effects of egg yolk on acrosome status before and after freezing were studied. Egg yolk significantly decreased the proportion of intact acrosomes before freezing, leading to fewer (P<0.05) intact acrosomes post-thaw and lower (P<0.05) recovery rates for intact acrosomes. In Experiment 3, including sodium dodecyl sulfate (SDS) in a diluent containing egg yolk tended to preserve the acrosome compared with the egg yolk containing diluent free of SDS, however, spermatozoa had a lower (P<0.05) proportion of intact acrosomes than those in a yolk-free diluent. However, after cooling, spermatozoa were diluted with a glycerolated extender containing egg yolk. Therefore, the objective of Experiment 4 was to explore whether the egg yolk or glycerol was responsible for the reduced intact acrosome percentage. In this experiment, after cooling and washing the spermatozoa were diluted in TCG with glycerol and/or egg yolk. The combination of glycerol and egg yolk in the extender reduced (P<0.05) the proportion of intact acrosomes compared with egg yolk or glycerol alone. In conclusion, the inclusion of egg yolk significantly improved sperm motility, indicating its beneficial effects during the freezing step of cryopreservation; trehalose appeared to synergistically increase its cryoprotective effects. Furthermore, although neither glycerol nor egg yolk per se affected the proportion of intact acrosomes, the combination of the two significantly reduced the proportion of acrosome-intact spermatozoa.     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.     

An evaluation of cryoprotective compounds on bovine spermatozoa

The potentially cryoprotective properties of 72 higher-molecular-weight polymeric additives and 69 low-molecular-weight compounds were evaluated. The polymeric compound selection was based on solubility in semen extender, toxicity and finally, on the cryoprotective effect on bull spermatozoa as measured by progressive motility. Five compounds showed cryoprotection to the cell, but with no significant improvement over that of TESNaK yolk, TEST yolk, or TEST yolk glycerol extenders used as controls. Low-molecular-weight compounds were selected on the basis of colligative properties particularly that of freezing-point depression. Elimination was based on precipitation of proteins in the extender, toxicity, and cryoprotection to bovine spermatozoa as measured by progressive motility. Nineteen compounds that yielded protection during cryopreservation of bovine spermatozoa were compared using post-thaw motility and membrane integrity using glutamic-oxaloacetic transaminase enzyme retained in the spermatozoa after freezing as an indicator. Semen was diluted with extender containing selected compounds at 35 or 5 °C to determine the effect of temperature at which the cryoprotective compound was added. Glycerol yielded the highest recovery. Diethylene glycol, dimethylsulfoxide, N-methylacetamide, and triethylene glycol appeared not to be different in freezing bovine spermatozoa. The temperature or method of addition of cryoprotective compound did not reveal a significant difference.     

Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen

Hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders in order to protect the spermatozoa against cold shock. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). In recent years, arguments concerning the presence of cryoprotective antagonists in egg yolk, have reinforced interest in the use of only the LDL extracted from egg yolk in the extenders. However, current methods of LDL purification do not support the use of LDL in commercial extenders because they offer a poor recovery rate. Consequently, we have developed an easy method to extract LDL from egg yolk. Several concentrations of purified LDL (between 2.5 and 20%, w/v) were tested in freezing extenders for bull semen, and compared with commercial extenders. Our extraction method reached 97% purity and about 67% yield, and is easily reproducible on an industrial scale. Analysis of sperm motility showed that the motility and characteristics of spermatozoa movement were improved with LDL in the extender, as compared to a commercial extender containing egg yolk. The optimum LDL concentration in the extender was 8%. In conclusion, we propose that an extender containing LDL extracted from egg yolk could be used as cryoprotective media with a better efficiency than present commercial extenders.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of egg yolk, glycerol and cooling rate

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.     

Freezing of stored, chilled dog spermatozoa

The aims of this study were to find out if dog spermatozoa can be stored chilled for 1 or 2 days prior to freezing without a deterioration in post-thaw vitality and longevity, and to compare two extenders; the Uppsala Equex-2 (UE-2) and a TRIS egg yolk extender (EYT). Pooled dog semen was frozen immediately after collection, or was extended and stored at 4 degrees C for 1 or 2 days before freezing. Sperm motility and acrosome integrity were evaluated before freezing and for 6h post thaw at 38 degrees C, while sperm plasma membrane integrity was evaluated post thaw. There were no effects of pre-freeze storage time or extender on post-thaw motility or plasma membrane integrity, but a significant effect of extender (P < 0.0153) on post-thaw acrosomal integrity was found, UE-2 being better than EYT. There was a significant (P < 0.0001) negative effect of post-thaw storage time on acrosome integrity, but this was not influenced by pre-freeze storage time or extender. In conclusion, we found that dog spermatozoa can be frozen after 1 or 2 days of cold storage without significant deterioration in post-thaw motility, acrosome integrity or sperm plasma membrane integrity compared to when frozen immediately after collection. The UE-2 extender was superior to the EYT extender for freezing of cold stored dog spermatozoa.     

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing-thawing caused a significant increase (P<0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P<0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing-thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P<0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P<0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.     

Effects of Equex STM Paste on the quality of frozen-thawed epididymal dog spermatozoa

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.     

Influence of extender,temperature, and addition of glycerol on post-thaw sperm parameters in ram semen

Using a two-step extension methodology, two experiments were conducted using a split-sample design to compare the effect on post-thaw ram sperm parameters of a milk-based extender (Experiment 1) containing four different egg yolk concentrations (5% [M5], 10% [M10], 15% [M15], and 20% [M20]), and a commercially available extender (Bioexcell); IMV, L'Aigle, France) free from additives of animal origin, containing two different final glycerol concentrations (3.2% [B] and 6.4% [BB]) (Experiment 2). In both experiments, glycerol was added either at 5 degrees C or at 15 degrees C together with the second fraction of each extender. The sperm characteristics assessed were motility (measured subjectively [SM] and by means of cell motion analysis (CASA), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline (CTC)/EthD-1). Results of Experiment 1 showed no significant positive effect of increasing the concentration of egg yolk above 10% on post-thaw motility, membrane integrity, or induction of sperm capacitation-like changes. In Experiment 2, Bioexcell (BB) yielded similar post-thaw results as did the milk extender (control). In both experiments, post-thaw sperm parameters were better preserved when glycerol was added at 5 degrees C, although the results were not always statistically significant for all variables studied. In conclusion, when using milk-based extenders for freezing ram semen, low (5-10%) concentrations of egg yolk and the addition of glycerol at 5 degrees C are recommended. Furthermore, the results indicate that when freezing ram semen, Bioexcell containing 6.4% glycerol may be used as an alternative extender to the conventional milk extender containing 5% egg yolk.     

Long-term preservation of chilled canine semen: effect of commercial and laboratory prepared extenders

The present study was conducted to evaluate chilled semen conservation over time in 3 commercial and 4 laboratory prepared extenders, including a new Tris-glucose extender. The beneficial effect of adding egg yolk to these media was also analyzed. The effects of these extenders on motility and acrosome reaction were characterized objectively using a computer-aided semen analyzer and the chlortetracycline staining, respectively. No significant differences were observed when comparing the different commercial extenders without egg yolk, but addition of egg yolk improved all motility parameters significantly (preservation of 50% of motility was observed at 3.2+/-1, 2.9+/-0.5, 2.3+/-0.5, 8.5+/-0.2, 5.4+/-1.1, 5.2+/-0.4 d, for Biladyl, green extender and fresh-phos extenders without and with egg yolk, respectively). Motility parameters were best preserved in egg yolk supplemented Biladyl extender with a mean percentage of 86.3+/-10.5 motile spermatozoa after 7 d at 4 degrees C. Efficacy of egg yolk-supplemented commercial extenders on sperm motility at 4 degrees C was (in decreasing order) as follows: Biladyl > green extender > fresh-phos. However, high quality motility and the percentage of motile spermatozoa were highest with some of the laboratory prepared extenders: a 50% conservation rate of motile spermatozoa was observed following the use of supplemented egg yolk extenders. These are classified in decreasing order as follows: Tris-glucose (13+/-1 d) > Tris-fructose (9.7+/-0.6) > EDTA (4.+/-0.6 d) > Tris-bes (3.6+/-1.1 d). A low concentration of motile spermatozoa was still observed in the Tris-glucose egg yolk extender 16 d after collection, clearly demonstrating the importance of the medium and the beneficial effect of egg yolk on sperm motility of 4 degrees C chilled semen. Similar effects of extender were observed for acrosome reactions. Egg yolk clearly had a protective effect reducing acrosome reactions significantly in all media tested as follows: the highest acrosome losses were observed in the fresh-phos and EDTA extenders without egg yolk; the lowest rate was observed with Tris-glucose supplemented egg yolk extender. In conclusion, at 4 degrees C, egg yolk extender best-protected sperm motility parameters. Differences in osmolarity between the extenders in terms of substrate related to sperm metabolic activity may explain the optimal results obtained using egg yolk-supplemented Tris-glucose extender, which preserved motility and acrosome integrity in chilled dog semen. These results indicated that good quality dog spermatozoa could be preserved for up to 10 d.     

Post-thaw survival of ram spermatozoa and fertility after insemination as affected by prefreezing sperm concentration and extender composition

A study was conducted to investigate the effects of prefreezing sperm concentration using two extenders on post-thaw survival and acrosomal status of ram spermatozoa (Experiment 1) and fertility after intrauterine insemination with differing doses of semen (Experiment 2). In autumn (Northern hemisphere), semen was collected by artificial vagina from 8 adult Leccese rams and ejaculates of good quality semen were pooled. Two extender systems for cryopreservation were considered, one based on milk-lactose egg yolk (Milk-LY) and the other based on tris-fructose egg yolk (Tris-FY). Experiment 1 (2 x 6 factorial scheme) examined the in vitro characteristics of spermatozoa in relation to the Milk-LY and Tris-FY extenders and six prefreezing sperm concentrations (50, 100, 200, 400, 500 and 800 x 10(6) spermatozoa/mL). Experiment 2 (2 x 4 factorial) evaluated the influence of the Milk-LY vs Tris-FY extenders and four doses (20, 40, 80 and 160 x 10(6) spermatozoa/0.25 mL) corresponding to prefreezing spermatozoa concentrations of 100, 200, 400 and 800 x 10(6) spermatozoa/mL, on fertility of ewes inseminated in uterus by laparoscope. Prefreezing sperm concentration influenced (P < 0.01) freezability of spermatozoa and affected negatively all the in vitro parameters at 800 x 10(6) spermatozoa/mL. Overall, Milk-LY tended to ensure higher viability and acrosomal integrity of spermatozoa after thawing at the intermediate sperm densities (range 100 to 500 x 10(6) spermatozoa/mL). At 500 x 10(6) spermatozoa/mL concentration corresponded the best condition for survival of spermatozoa (71.2%), acrosome integrity (71.5%) and acrosomal loss (6.0%). At the lowest sperm concentration (50 x 10(6) spermatozoa/mL), Tris-FY resulted in a higher survival rate than Milk-LY (61.3%, P < 0.05) and lower acrosomal loss (9.7%, P < 0.05). Milk-LY supported spermatozoa motility better than Tris-FY after incubation at sperm concentration between 50 and 400 x 10(6) spermatozoa/mL (0.05 > P < 0.01). Semen doses of 20 to 40 x 10(6) spermatozoa/ewe provided satisfactory fertility rates (64 to 81%). The increase of inseminate doses to 160 x 10(6) spermatozoa/ewe failed to improve fertility, actually tending to decrease lambing rates.     

Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders

Ten semen extenders were tested in two experiments for cryopreservation of semen collected from four Finn and four Dorset rams. Two ejaculates of semen were combined from each ram for testing each extender treatment. The extenders consisted of a series of commonly used egg yolk-TRIS media with and without sodium and triethanolamine lauryl sulfate (STLS), a similar extender with 3-N-morpholino propane sulfonic acid (MOPS), and milk and whey extenders. In Experiment 1, extender treatments were replicated with three sets of collections from the eight rams, and in Experiment 2 with two sets. The egg yolk-TRIS-glycerol-STLS (EY(1)TSTLS) extender was significantly superior to other extenders except whole milk in protecting the sperm during freezing and thawing. In Experiment 1, a 20% egg yolk-TRIS-glycerol-STLS extender preserved 71% of the progressively motile Finn sperm (post-thaw divided by pre-freeze percentage of motile sperm), and 76% of the Dorset sperm. In Experiment 2, the corresponding values for the same EY(1)TSTLS extender used with Finn and Dorset sperm were 86 and 64%, respectively. Without STLS the egg yolk extenders were significantly less effective in protecting cryopreserved ram sperm. This egg yolk-TRIS extender, containing STLS and glycerol, may hold promise for freezing ram sperm that could be used successfully for intracervical insemination.     

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

The cryoprotective effect of low-density lipoproteins in extenders on bull spermatozoa following freezing–thawing

Egg low-density lipoprotein (LDL) was added at concentrations of 7–10% to the extenders used to freeze bull semen and its effects on the motility, mitochondria activity, acrosome integrity, membrane integrity and DNA integrity of frozen–thawed sperm were assessed. Analysis of data showed that the motility and characteristics of spermatozoa movement were higher with LDL in the extender, as compared to the extender containing 20% egg yolk. The results indicated that 8% LDL supplementation provided the highest sperm motility (55.8%) and movement characteristics (VSL, straight linear velocity: 33.8 μm/s; VCL, curvilinear velocity: 50.2 μm/s; LIN, linearity index: 56.5%; STR, mean coefficient: 76.7%; VAP, average path velocity: 35.9 μm/s; WOB, wobble coefficient: 63.9%). A concentration of 10% LDL resulted in a significant decline in the VSL, LIN, VAP and WOB values (P < 0.05). Supplementation of LDL at 8% LDL resulted in significantly higher spermatozoa mitochondrial activity, acrosome integrity, membrane integrity and DNA integrity (P < 0.05). According to all measured parameters, the extender containing 8% LDL showed beneficial cryoprotective effects on frozen–thawed bull spermatozoa. In conclusion, our results indicated that the extender containing 8% LDL extracted from egg yolk could be used successfully in the cryopreservation of bull semen with an efficacy that would be greater than present extenders containing 20% egg yolk.     

Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezing-thawing?

In this study a radioisotope method, which is based on the quantitative measurements of tritiated-labeled actinomycin D ((3)H-AMD) incorporation into the sperm nuclei ((3)H-AMD incorporation assay), was used to assess the chromatin status of frozen-thawed boar spermatozoa. This study also tested the hypothesis that frozen-thawed spermatozoa with altered chromatin were susceptible to DNA fragmentation measured with the neutral comet assay (NCA). Boar semen was diluted in lactose-hen egg yolk-glycerol extender (L-HEY) or lactose ostrich egg yolk lipoprotein fractions-glycerol extender (L-LPFo), packaged into aluminum tubes or plastic straws and frozen in a controlled programmable freezer. In Experiment 1, the chromatin status and DNA fragmentation were measured in fresh and frozen-thawed spermatozoa from the same ejaculates. There was a significant increase in sperm chromatin destabilization and DNA fragmentation in frozen-thawed semen as compared with fresh semen. The proportions of spermatozoa labeled with (3)H-AMD were concurrent with elevated levels of sperm DNA fragmentation in K-3 extender, without cryoprotective substances, compared with L-HEY or L-LPFo extender. Regression analysis revealed that the results of the (3)H-AMD incorporation assay and NCA for frozen-thawed spermatozoa were correlated. Boars differed significantly in terms of post-thaw sperm DNA damage. In Experiment 2, the susceptibility of sperm chromatin to decondensation was assessed using a low concentration of heparin. Treatment of frozen-thawed spermatozoa with heparin revealed enhanced (3)H-AMD binding, suggesting nuclear chromatin decondensation. The deterioration in post-thaw sperm viability, such as motility, mitochondrial function and plasma membrane integrity, was concurrent with increased chromatin instability and DNA fragmentation. This is the first report to show that freezing-thawing procedure facilitated destabilization in the chromatin structure of boar spermatozoa, resulting in an unstable DNA that was highly susceptible to fragmentation.     

Effect of butylated hydroxytoluene on cryopreservation of Boer goat semen in Tris egg yolk extender

The aim of this study was to determine the effect of butylated hydroxytoluene (BHT), a lipid-soluble anti-oxidant added in different concentrations to the Tris egg yolk extenders on semen cytological parameters pre freezing and post thawing (motility, morphology, viability, acrosome integrity and membrane integrity) of Boer goat spermatozoa. A total of 40 ejaculates from four Boer goat bucks were collected using an artificial vagina. Ten replicates of the ejaculates were diluted with a Tris egg yolk based extender which contained various concentrations (0.5mM, 1.0mM, 2.0mM and 3.0mM) of butylated hydroxytoluene while one sample was processed without supplementation of antioxidant and served as control. The diluted semen was cooled at 4°C and loaded into the straw and then stored in liquid nitrogen. It was evident that supplementation of BHT produces positive effect in terms of motility, membrane integrity and acrosome integrity in comparison with the control group in cooled and frozen Boer goat semen. Results showed significant differences in motility, membrane integrity, acrosome integrity and viability of cooled and frozen Boer goat spermatozoa at different concentrations. Motility, membrane integrity, acrosome integrity and viability was significantly higher in all treated groups than the control group (P<0.05) while there was no significant differences (P>0.05) in morphology trait between all group in cooled semen. However, improvement (P<0.05) was observed only in terms of the membrane integrity and acrosome integrity compared to the control and other treated groups in frozen semen. In conclusion, BHT can be used in cryopreservation of Boer goat semen in order to reduce the oxidative stress on spermatozoa.     

Trehalose improves rabbit sperm quality during cryopreservation

High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, acrosome integriy, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry.     

Effect of seminal extenders containing egg yolk and glycerol on motion characteristics and fertility of stallion spermatozoa

Three experiments were conducted to evaluate the effects of egg yolk and(or) glycerol added to a nonfat dried skim milk-glucose (NDSMG) extender on motion characteristics and fertility of stallion spermatozoa. In Experiment 1, ejaculates from each of 8 stallions were exposed to each of 4 extender treatments: 1) NDSMG, 2) NDSMG + 4% egg yolk (EY), 3) NDSMG + 4% glycerol (GL), and 4) NDSMG + 4% egg yolk + 4% glycerol (EY + GL). Samples were cooled at -0.7 degrees C/min from 37 to 20 degrees C; subsamples were then cooled at -0.05 or -0.5 degrees C/min from 20 to 5 degrees C. Percentages of motile spermatozoa (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, 24 and 48 h after initiation of cooling. There was no overall effect (P > 0.05) of cooling rate. PMOT was highest (P < 0.05) for spermatozoa extended in NDSMG + GL at 48 h. At 24 and 48 h, MOT and PMOT were lowest (P < 0.05) for spermatozoa extended in NDSMG + EY. In Experiment 2, ejaculates from 8 stallions were exposed to each of 4 treatments: 1) NDSMG, 2) NDSMG + EY, 3) semen centrifuged in NDSMG and resuspended in NDSMG, and 4) semen centrifuged in NDSMG and resuspended in NDSMG + EY. Samples were cooled from 20 to 5 degrees C at each of 2 rates (-0.05, -0.5 degrees C/min). A detrimental interaction between seminal plasma and egg yolk was noted for PMOT at 6 h and for both MOT and PMOT at > or = 24 h postcooling. Experiment 3 determined if egg yolk or glycerol affected fertility. The seminal treatments were 1) NDSMG, 2) NDSMG + EY with previous removal of seminal plasma, and 3) NDSMG + GL. All samples were cooled to 5 degrees C and stored 24 h before insemination. Embryo recovery rates 7 d after ovulation were lower for mares inseminated with spermatozoa cooled in NDSMG + EY (17%, 4/24) or NDSMG + GL (13%, 3/24) extenders, than semen cooled in NDSMG (50%, 12/24). We concluded that egg yolk (with seminal plasma removal) or glycerol added to NDSMG extender did not depress MOT or PMOT of cooled stallion spermatozoa but adversely affected fertility.