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.     

Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS

Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP-106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.     

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10⁵ spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p < 0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p < 0.05).     

Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa

The aim of this study was to evaluate different antioxidants-supplemented freeze-dried egg yolk based extenders for the post-thawing quality and incubation resilience of goat spermatozoa. Pooled semen were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in control and antoxidant supplemented freeze-dried extenders (methionine, cysteamine and butylated hydroxytoluene). Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Membrane lipid peroxidation status was also analyzed using the malondialdehyde (MDA) concentration. In the study, antioxidant supplemented freeze-dried egg yolk based extenders have beneficial effect on goat sperm parameters. In addition, we achieved a higher quality in post thawed goat semen even after 6 h incubation when the extender was supplemented by 5 mM BHT or cysteamine.     

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.     

Influence of glucose and fructose in the extender during long-term storage of chilled canine semen

The use of chilled, extended semen in dog breeding is becoming increasingly popular as preparation and transportation is less expensive and regulations are often less complicated than for frozen semen. Sugar is one of the main constituents in semen extenders, and glucose and fructose are metabolized in separate pathways by freshly ejaculated dog sperm. In this study, glucose, fructose or an equal mixture of both were used in an egg-yolk-tris (EYT) extender at two different concentrations (10 and 70 mM). EYT extender without sugar supplementation, providing only the glucose (3-4 mM) originating from the egg-yolk, served as a control. The longevity of the chilled semen at 5 degrees C was 23 days: the quality of physical and functional characteristics decreasing with time. Glucose and fructose had a strong influence on motility and movement patterns of chilled canine semen. The beneficial effect of 70 mM sugar concentrations compared to 10 mM and the control was pronounced, and maintained sperm motility > or = 70% for 8 days of storage, compared to for 4 days in the control extender. Fructose maintained higher sperm motility than did glucose and the mixture. VAP values were higher in sugar-supplemented extenders (P < 0.05). Neither type nor concentration of the two sugars influenced sperm plasma membrane, acrosome integrity or the acrosome reaction following ionophore challenge (ARIC). Sugar consumption by dog sperm varied between the different periods of storage and with sugar concentrations provided in the extenders. Glucose consumption by dog sperm was greater than fructose consumption when both sugars were present in equal amounts, indicating that dog sperm used glucose in preference to fructose. In conclusion, the major influence of the two sugars on chilled semen was to support motility. EYT extender supplemented with fructose at a concentration of 70 mM was found to be the best of the tested extenders for long-term preservation of chilled canine semen.     

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%).     

Incorporation of taurine and hypotaurine did not improve the efficiency of the Uppsala Equex II extender for dog semen freezing

The working hypothesis of the present study was that supplementation of the Uppsala Equex II (UE) extender with the amino acid (AA), taurine (T) and hypotaurine (H) would improve dog sperm post-thaw quality, as previously seen for ram and bull semen, respectively. Five pools from 15 ejaculates of 15 dogs were used. Each AA was added to the UE extender at a concentration of 25, 50 and 7 5mM. Amino acid-free extender was used as a control. The following post-thaw parameters were evaluated: sperm motility by light microscopy and by CASA evaluation, longevity, viability (eosin-nigrosin staining), and flow cytometry (FC) was used to assess acrosome integrity and mitochondrial activity after PI/Fitc-PSA and PI-Rhodamine staining, respectively. Post-thaw sperm motility and velocity did not differ among extenders. Amplitude of lateral head displacement was lower for sperm frozen in the 25 mM H-supplemented extender. Semen frozen in the extender with 50 mM of T resulted in higher number of live sperm with damaged acrosomes after thawing. Higher numbers of live sperm with minimal mitochondrial activity were obtained for samples frozen with 25 and 50 mM T-supplemented extenders. Semen frozen in the control and 50 mM T-supplemented extenders had the highest number of live (eosin-nigrosin stain negative) sperm immediately post-thawing. We concluded that supplementation of the Uppsala extender with T or H did not improve sperm post-thaw mitochondrial activity or semen motility and viability.     

Effects of supplementation of Tris-egg yolk extender with royal jelly on chilled and frozen-thawed ram semen characteristics

This study was conducted to examine the effect of supplementation of Tris-egg yolk extender with lyophilized royal jelly (RJ) on chilled and frozen-thawed ram semen parameters. Ejaculates were collected by artificial vagina from 4 mature rams, twice a week for 4 weeks. Only samples with motility of ≥70% were included, pooled and divided into four equal parts and then diluted in extenders with various concentrations of RJ (0, 1, 3 and 5%, vol/vol) to a final concentration of 200 × 10⁶ sperm/mL and was incubated at 37 °C for 30 min and were subsequently evaluated. After equilibration of extended semen for 2 h at 4 °C, some semen samples were packed in 0.25 mL plastic straws. Then, the straws were frozen in the liquid nitrogen vapor phase for 15 min and stored at −196 °C in liquid nitrogen. The frozen straws were thawed in warm water (37 °C) for 30 s and evaluated; whereas, other semen samples were stored in the refrigerator (4 °C) up to 7 days. The chilled samples were kept in water bath (37 °C) for 5 min and then were evaluated. After dilution, the lowest and highest sperm total abnormality was recorded in 3 and 5% RJ supplemented groups, respectively (P < 0.05). The chilled sperm total motility and membrane integrity were significantly (P < 0.05) higher in 3% than those in 0% and 5% RJ supplemented groups. The chilled sperm progressive motility and viability was significantly (P < 0.05) higher in 1 and 3% than those in 0 and 5% RJ supplemented groups. The frozen-thawed sperm total motility, progressive motility, membrane integrity and viability were significantly higher in 3% RJ supplemented group (P < 0.05). In conclusion, supplementation of Tris-egg yolk extender with 3% lyophilized RJ had a protective effect on chilled and cryopreserved ram spermatozoa.     

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.     

Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: Comparison between cysteine and rosemary (Rosmarinus officinalis)

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10 mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3 h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10 mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3 h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system.     

Effect of low density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen

This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 10⁶ motile spermatozoa mL⁻¹) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (-196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa.     

Evaluation of extenders and cryopreservatives for cooling and cryopreservation of spermatozoa from the western spotted skunk (Spilogale gracilis)

Experiments were conducted to develop a suitable protocol for cryopreservation of spotted skunk semen. Semen was collected by electroejaculation of captive male skunks (n = 16) from late January through late November. In the first experiment, fresh semen was diluted in either TEST (n = 10), TRIS (n = 9), or BF5F (n = 7) extenders and maintained at 4°C for 16 hr. Sperm motility in these extenders was not significantly different before cooling (P = 0.71), but samples diluted with BF5F exhibited significantly lower sperm motility than the other extenders at all time points after cooling (P < 0.05). In the second experiment, fresh semen was diluted in TEST containing either 3, 5, or 10% DMSO or 3, 5, or 10% glycerol as a cryopreservative. These samples were cooled to 4°C and frozen in 0.25 ml French straws on dry ice. Some samples containing 5% DMSO or 5% glycerol (n = 4), were also frozen on dry ice as pellets. Frozen samples were maintained in liquid nitrogen. Fresh samples had significantly greater sperm motility in dimethyl sulfoxide (DMSO) than in glycerol (P < 0.05), while frozen and thawed samples had the highest motility in 5 or 10% DMSO or 10% glycerol. Samples frozen in French straws had significantly greater sperm motility after freezing and thawing than those frozen by the pellet method (P < 0.05). Optimum cryoprotection was achieved with the TEST extender containing 5 or 10% DMSO, when used in conjunction with French straws. © 1992 Wiley-Liss, Inc.     

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca)

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ± 7.7%) and membrane functionality (60.5 ± 4.2%) and higher mitochondrial activity (21.5 ± 3.7%) than those preserved in ACP-117c (20.9 ± 5.4% motile sperm; 47.1 ± 2.5% functional membrane; 11.8 ± 1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ± 3.3%) in comparison to samples diluted in ACP-117c (18.6 ± 1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.     

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.     

Comparing ethylene glycol with glycerol for cryopreservation of buffalo bull semen in egg-yolk containing extenders

The objective of this work was to evaluate the possibility of substituting glycerol for ethylene glycol when cryopreserving buffalo semen. Semen of eight buffalo bulls was mixed, pooled, and frozen in one of these four diluents: centrifuged Tris egg yolk glycerol; centrifuged Tris egg yolk ethylene glycol; centrifuged Milk egg yolk glycerol; or centrifuged Milk egg yolk ethylene glycol. Semen quality parameters assessed after thawing were motility, survivability, livability, sperm abnormality, acrosome integrity, and plasma membrane integrity. Conception rate and pregnancy rate were calculated after insemination of 104 buffaloes by straws of different groups (26 female/extender). Improvement in livability, sperm abnormality, acrosome integrity, plasma membrane integrity, conception rate, and pregnancy rate were seen when using ethylene glycol to replace glycerol when freezing buffalo bull semen in centrifuged TRIS egg yolk 61.15 ± 0.73, 24.85 ± 0.41, 69.10 ± 0.81, 71.75 ± 0.72, 46.2%, and 46.2%, respectively, followed by centrifuged milk egg yolk extenders.     

Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen

This study was performed to evaluate the effectiveness of quercetin as a non-enzymatic antioxidant in combination with glycerol or Dimethylacetamide (DMA), on freezability of goat semen. Ejaculates from four healthy mature Mahabadi goats were collected using an artificial vagina. After primary processing, semen was pooled and extended by egg yolk based extender supplemented with different concentrations of quercetin (10 or 20 μM) along with 5% glycerol or DMA. The extended semen was frozen and sperm motility parameters, viability, abnormality, membrane integrity and lipid peroxidation were assessed after thawing. Results showed that sperm viability, total motility, progressive motility, straightness (STR) and linearity (LIN) were higher (P < 0.05), and abnormality percentage and MDA concentration were lower (P < 0.05) in extender containing DMA. Similarly, higher (P < 0.05) total motility, progressive motility, viability and membrane integrity along with lower (P < 0.05) MDA level were noted in Q10 group. The lowest (P < 0.05) MDA level was observed in DMA extender containing moderate level of quercetin (Q10D). Also the STR was higher (P < 0.05) in Q10D compared to Q10G and Q20G groups. In conclusion, supplementation of extender with 10 μM quercetin in combination with DMA improves the goat sperm motion kinetics and suppresses lipid peroxidation after freezing and thawing. Furthermore, DMA is more effective cryoprotectant for the freezing of goat sperm.     

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer‐assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.     

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.     

Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits

This study was conducted to elucidate the effect of different freezing extenders on two lines selected for hyperprolificacy and longevity (H and LP, respectively). In extender A, dimethyl sulphoxide (Me₂SO) and sucrose were used as cryoprotectants. In extenders B and C, the sucrose was replaced by 20% egg yolk, and in extender C the Me₂SO was substituted by acetamide. Semen was packaged in 0.25 ml plastic straws and cooled at 5°C for 45 min, and then was frozen in liquid nitrogen vapour for 10 min before being plunged into the liquid nitrogen. Thawing was carried out by immersing the straws in a water bath at 50°C for 10 s. Frozen-thawed semen characteristics and reproductive parameters were affected by freezing. Extender C showed significantly lower post-thawing quality traits than any of the three extenders. Acrosome integrity was significantly improved when Me₂SO was used as cryoprotectant. Sucrose replacement by 20% egg yolk had no effect on acrosome integrity but provided significantly lower sperm motility and viability. Freezing extender affected fertility rate, total born, number of implantation sites and gestational losses, obtaining better results when extender A was used. The acrosomal integrity after frozen-thawed process showed a significant correlation with fertility at 12^th day and also at birth, indicating that an increase in acrosomal integrity leads to an increase in both fertilities (12^th day and at birth). A positive correlation between motility of semen and implantation sites was found. The post-thawing quality traits of semen were not affected by the genetic line, although LP line showed higher total born and lower foetal and gestational losses. The findings of this study suggest that freezing extender composition has a significant effect on the success of rabbit sperm for preservation, and when Me₂SO was used as permeable cryoprotectant sucrose provided better protection compared with egg yolk and improved reproductive traits, and, on the other hand, the male genotypes used in the present study had no effect on frozen-thawed sperm parameters but negatively affected some of the reproductive parameters.