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.     

Trehalose enhanced the freezability of Poodle dog sperm collected by an artificial vagina (AV)

In an attempt to develop a suitable freezing method for Poodle dog sperm, an experiment was conducted to investigate semen collection methods of digital stimulation and an artificial vagina (AV), using Tris and trehalose-egg yolk extender, on the characteristics and cryopreservation of sperm. Two dogs (dogs A and B) were subjected to semen collection by digital stimulation and AV. The volume, sperm concentration, sperm motility index (SMI) and acrosome status of ejaculates were determined immediately after collection. The remainder was frozen as pellets in Tris and trehalose-egg yolk extender. Sperm motility index was evaluated after thawing and during a thermal resistance test, and acrosome integrity was also assessed. No significant differences regarding sperm concentrations, SMI and acrosome integrity were observed between semen collected by AV and digital stimulation. However, when dog sperm were collected by an AV and frozen in trehalose-egg yolk extender, the motility index of frozen-thawed sperm was significantly improved compared to sperm frozen in Tris-egg yolk extender which were collected by digital stimulation. In conclusion, semen collected by an AV and frozen in trehalose-egg yolk extender was effective in enhancing the freezability of Poodle dog sperm.     

Trehalose-enhanced fluidity of the goat sperm membrane and its protection during freezing

In an attempt to find a suitable freezing method for goat semen, two experiments were conducted to study the influence of trehalose on the cryopreservation of goat spermatozoa. In experiment 1, goat spermatozoa were frozen in trehalose extender (0.375 M) alone (100%) or at different combinations of trehalose with Tris-citric acid-glucose (TCG) extender (0%, 25%, 50%, 75%). Final concentrations of 20% (v:v) egg yolk and 4% (v:v) glycerol were employed in the extenders (osmolality = 370, pH = 7). Sperm motility was assessed using a computer-assisted sperm analysis system and acrosome integrity was assessed using fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA). The sperm-motility parameters improved significantly by increasing the concentration of trehalose (P < 0.05) and significantly high recovery rates for the motility parameters were also achieved by a high concentration of trehalose (P < 0.05). Motility of the frozen-thawed spermatozoa after a 3-h incubation improved significantly with increasing concentrations of trehalose in the extender (P < 0.05). The 75% and 100% trehalose extenders yielded a significant increase in the percentage of spermatozoa with intact acrosome (P < 0.05). In experiment 2, merocyanine 540/Yo-Pro staining was used to study the influence of trehalose on membrane fluidity compared with that of sucrose and TCG. Percentage of cells with high merocyanine fluorescence was significantly higher in spermatozoa treated with trehalose than sucrose or TCG (P < 0.05), indicating a significantly highest membrane fluidity of sperm samples extended with trehalose solution. We thus conclude that the substitution of a Tris-citric acid diluent composition with trehalose significantly improves the freezability of goat spermatozoa. Furthermore, the cryoprotective effects of trehalose observed in this study may be due to enhanced sperm membrane fluidity before freezing.     

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.     

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.     

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.     

Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs–Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.     

Fertility of spermatozoa cryopreserved with 2% acetamide or glycerol through artificial insemination in the Japanese white rabbit.

The rabbit is considered to be a valuable laboratory animal. We compared 2% acetamide and glycerol as cryoprotectants in egg-yolk diluent for ejaculated Japanese white rabbit spermatozoa to improve sperm cryopreservation methods. Fertility through artificial insemination, forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rates of forward progressive motility and plasma membrane integrity of the spermatozoa frozen with acetamide (27.1 +/- 8.3% and 24.5 +/- 6.5%) were significantly (P < 0.05) higher than those of the spermatozoa frozen with glycerol (16.3 +/- 10.9% and 14.3 +/- 7.6%). Though there was no significant difference in the kindling rates, the litter size of females inseminated with spermatozoa frozen with acetamide (6.0 +/- 1.1) were significantly (P < 0.05) higher than those of spermatozoa frozen with glycerol (3.0 +/- 0.4). The results indicate that 2% acetamide has a higher cryoprotective effect than 2% glycerol for sperm cryopreservation in the Japanese white rabbit.     

Arbutin's suppression of cryodamage in goat sperm and its mechanism of cryoprotection

Arbutin (4-hydroxyphenyl-glucopyranoside) is a glycosylated hydroquinone present in high concentrations in the leaves of several plants capable of surviving prolonged, extreme dehydration. Two experiments were conducted to determine the effects of arbutin on cryopreservation of goat sperm. In Experiment 1, goat sperm were frozen in extenders with various ratios of Tris-citric acid-glucose (TCG) and arbutin; concentrations of the latter were 0.0 (only TCG), 0.1, 0.2, 0.3, and 0.4 M (only arbutin)]. All extenders had 20% (v/v) egg yolk (EY) and 4% (v/v) glycerol (osmolality = 370 mOsm, pH = 7.0). Sperm motility and acrosome integrity were assessed using CASA, and fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA), respectively. Percentages of motile and progressively motile sperm improved with the addition of arbutin; results were optimal (89.0 and 70.0%, respectively; P < 0.05), with 0.4 M arbutin. Furthermore, arbutin improved (P < 0.05) post-thaw recovery rates for both motility and progressive motility. After incubation for 3 h, motility of frozen-thawed washed sperm improved (70%, P < 0.05) with arbutin in the extender. The percentage of sperm with an intact acrosome peaked (77.2%, P < 0.05) with 0.4 M arbutin in the extender. In Experiment 2, the percentage of cells with merocyanine 540/Yo-Pro staining was higher in sperm treated with arbutin than with TCG (P < 0.05), with the best result (58.0%) with 0.4 M arbutin; therefore, arbutin increased membrane fluidity. In conclusion, substitution of a TCG-EY diluent composition with arbutin improved freezability of goat sperm (apparently due to increased membrane fluidity). Furthermore removal of arbutin by centrifugation after freezing and thawing increased sperm longevity.     

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.     

The effect of viscosity of semen diluents on motility of bull spermatozoa

The effect of egg yolk extender on semen viscosity and bull sperm motility of fresh and cooled or deep frozen semen was determined by a computer-assisted system. Viscosity of the extender was determined by flow time. Based on the sperm velocity (velocity of the average path), individual spermatozoon were classified into groups of progressively motile (>==30 microm/sec) and immotile (<10 microm/sec) spermatozoa. The average velocity of progressively motile spermatozoa (VPM), the velocity of linear progressively motile spermatozoa (VLP) and the percentage of linear swimming spermatozoa (LIN) were evaluated. The addition of 10, 20 or 30% egg yolk to Tris buffer (pH 6.5) resulted in a linear decrease of VPM and a decrease in the percentage of progressively motile spermatozoa, but it increased the relative rate of LIN in fresh diluted semen. Increasing the levels of egg yolk in the diluent resulted in higher viscosity. The VLP was significantly higher than the VPM. In refrigerated or frozen semen samples, extender with 30 and 20% egg yolk had a similar effect on the VPM but not on the percentage of progressively motile sperm cells. Freezing of egg yolk (30%) extender to -20 degrees C resulted in a significant increased flow time and higher viscosity. Dilution of semen samples with high viscosity extender decreased the VPM in fresh and chilled semen. Freezing semen of high viscosity extender with glycerol had no apparent effect on the percentage of progressively motile spermatozoa compared with that of non-glycerinated egg yolk extender. The results suggest that different concentrations of egg yolk in the extender can influence the parameters of semen viscosity and sperm motility evaluated by a computer-assisted system.     

Decrease in glutathione (GSH) content in bovine sperm after cryopreservation: comparison between two extenders

Growing evidence suggests that among the causes which deteriorate qualitative and functional characteristics of semen after freezing and thawing, there are those linked to oxidative stress and decrease detoxification of antioxidants present in the cells and seminal plasma. Overall, cryopreservation reduces the spermatozoal GSH content. In the present study we assessed the motility, capacitation status, ability to undergo acrosome reaction and levels of GSH in bovine spermatozoa after freezing and thawing, and compared the effects of the egg yolk tris-citrate and a commercial extenders (Bioxcell). Six semen samples were collected from each of five bulls and frozen and thawed in the two extenders. The level of total GSH of bovine spermatozoa detected in the raw semen ranged from 246.5 to 776.2 pmoles/mg of protein. The Bioxcell extender was superior in preserving GSH content (647.2+/-300.1 versus 223.6+/-154.0 pmoles/mL; P<0.05), motility levels (44.3+/-4.8 versus 41.8+/-4.0%; P<0.05), non-capacitated state (68.3+/-13.6 versus 61.3+/-17%; P<0.05) and a lower proportion of acrosome reacted spermatozoa post thaw (3.8+/-2.1 versus 6.3+/-3.1%; P<0.001) compared to egg yolk tris-citrate extender. These findings could be partially ascribed to the high GSH level contained in the commercial extender which seem able to alleviate oxidative damages to spermatozoa surviving freezing thawing procedures. Further researches are required in order to validate in vivo these results.     

Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing

Influence of different sugars supplemented to the extender on the motility, viability and intact acrosome rates of dog spermatozoa during dilution, equilibration and freezing was studied. The ejaculate was divided into 10 aliquots, which were diluted 1:3 with TRIS-citric acid extender containing 240 mMTRIS, 63 mM citric acid, 8% (v/v) glycerol, 20% (v/v) egg yolk and 70 mM sugar, which was either fructose, galactose, glucose, xylose (monosaccharide), lactose, trehalose, maltose, sucrose (disaccharide) or raffinose (trisaccharide). No sugar was added to the extender in the control group. Extended semen samples were cooled to 5 degrees C over 45 min, packaged in 0.25-mL straws, equilibrated for 2 h at 5 degrees C and frozen in liquid nitrogen vapor. Samples were thawed by placing straws into 37 degrees C water for 30 sec. Motility, viable sperm and intact acrosome rates decreased gradually in all groups after equilibration and consecutively freezing (P<0.001). The type of sugar significantly effected motility, viability and acrosomal integrity during equilibration and freezing (P<0.05). Galactose, lactose, trehalose, maltose and sucrose reduced damaged acrosome percentages in equilibrated samples (P<0.05). Sugar supplementation did not enhance motility and viability during equilibration. The disaccharides, except lactose, reduced post-thaw dead sperm and/or damaged acrosome percentages without promoting post-thaw motility (P<0.01), whereas monosaccharides, especially fructose and xylose, improved motility (P<0.05) along with viability and intact acrosome rates (P<0.05). Trehalose, xylose and fructose significantly increased total active sperm rates (motility x live sperm rate x normal acrosome rate) compared to other sugars (P<0.01) and control (P<0.0001) in frozen thawed samples. Therefore, sugar supplementation of the extender influenced post-equilibration and post-thaw sperm quality, and the type or locality of protective impact of the sugar on dog spermatozoa vary according to type of the sugar.     

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 basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa

The improvement of freezing extenders is critical when defining sperm cryopreservation protocols for wild species, in order to create germplasm banks. The aim of this study was to evaluate the effect of additives (Equex Paste and EDTA) supplementation, egg-yolk (10 and 20%) and glycerol (4 and 8%) concentrations and extender osmolality (300 and 320 mOsm/kg) on the post-thawing quality of brown bear semen. Semen was obtained from 20 adult males by electroejaculation, and centrifugated individually (600 × g for 6 min). The pellets were diluted 1:1 in the corresponding extender TTF (TES-Tris-Fructose with the aforementioned variants) and cooled to 5 °C. Then, it was diluted down to 100 × 10⁶ spz/mL, loaded in 0.25 mL straws and frozen at −20°C/min. After thawing (in water at 65 °C for 6s), the semen samples were assessed for motility (CASA), viability (SYBR-14 with propidium iodide), acrosomal status (PNA-FITC with propidium iodide) and mitochondrial activity (JC-1). Extender supplementation with additives rendered significantly higher results for these sperm parameters. Comparing the two percentages of egg yolk, 20% egg yolk showed the highest motility results, percentages of viable spermatozoa and viable spermatozoa with intact acrosome. No differences were detected among samples frozen using 4 or 8% glycerol. For extender osmolality, 300 mOsm/kg showed higher values of VAP, VCL, VSL, and ALH than 320 mOsm/kg. Based on the best performance of sperm motility, viability and acrosome status, we conclude that the most suitable extender to cryopreserve brown bear spermatozoa was TTF adjusted to 300 mOsm/kg, supplemented with 20% egg yolk, 4-8% glycerol, and the additives 1% Equex paste and 2% EDTA.     

Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri)

Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at −172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability.     

Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl, a commercial egg yolk extender

Low-density lipoproteins (LDL) have been previously isolated and identified as the cryoprotective fraction of yolk. The effect of LDL on sperm motility after freezing-thawing has been reported, but no study has been made to assess the effect of LDL on bull semen fertility. The aim of this study was to evaluate the fertility of bull semen cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Optidyl, a commercial extender containing egg yolk. To evaluate the fertilizing ability of semen, we used in vitro fertilization test, whereas acrosome and plasma membrane integrity were also evaluated. The percentage of motile spermatozoa was two fold higher after freezing in LDL than in Optidyl 54.4% versus 30.2% (P < 0.05). The cleavage rate was significantly higher after fertilization with semen frozen in LDL than with Optidyl 63.0% versus 54.8% (P < 0.05). No significant difference was observed on the blastocyst rate after in vitro culture. Integrity of the acrosome and the plasma membrane were maintained in both extenders. In conclusion, LDL preserve bull semen quality and fertilizing ability, allowing also better semen motility, after the freeze-thaw process.     

Cryopreservation of goat spermatozoa: comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination

TRIS-glucose or skim milk extenders are most commonly used for cryopreserving goat sperm. The aim of this study was to compare the ability of two extenders based on TRIS and skimmed milk buffer to maintain sperm viability after cryopreservation. Goat semen samples (n=110) were frozen with TRIS and with milk extender and thaw. Sperm motion parameters, morphology and acrosomal integrity were assessed in fresh and frozen-thawed samples by Sperm Class Analyzer (SCA) and Diff-Quik and Spermac staining techniques. Pregnancy rates were obtained after cervical insemination with frozen semen doses. The cryopreservation process had a significant effect on acrosome and kinematic parameters. TRIS extender provided more effective preservation of total motility, velocity parameters and amplitude of lateral head displacement after freezing. The percentage of acrosome intact spermatozoa was significantly higher in samples diluted with milk extender. In the insemination doses, mean values of velocity parameters and lateral head displacement were higher in doses processed in TRIS. Spermatozoa frozen in milk extender was mathematically greater than for those frozen with TRIS extenders, though no significant difference exists. We conclude that post-thaw kinematic parameters and acrosome integrity assessed after 1h of incubation was acceptable in both extenders which indicated the feasibility of cryopreserving goat spermatozoa. TRIS extender results in better in vitro performance compared to milk, though these improvements were not reflected in fertility results. Semen doses cryopreserved in milk extender provided greater pregnancy rates after intra-cervical insemination compared to those in TRIS extender (52.4% versus 42.9%).     

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.