Usefulness of powdered and fresh egg yolk for cryopreservation of Zebu bull spermatozoa

This experiment was designed to compare powdered egg yolk with fresh egg yolk in an extender for cryopreservation of Zebu bull semen. Sperm motility, plasma membrane integrity and viability were assessed at different stages of cryopreservation (post-dilution, pre-freezing and post-thawing). Sperm plasma membrane integrity remained similar at all the stages of cryopreservation. Sperm motility and viability were significantly higher after thawing in the extender containing powdered egg yolk. In conclusion, powdered egg yolk may be used in an extender for the cryopreservation of Zebu bull spermatozoa.     

Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender

This study was designed to compare commercially available extender Bioxcell^® with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 °C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 °C having 50 × 10⁶ spermatozoa/ml) in tris-citric egg yolk or Bioxcell^® extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, semen straws were thawed at 37 °C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell^® as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 ± 1.1, 45.0 ± 1.4), viability (66.2 ± 1.1, 64.4 ± 1.3) plasma membrane integrity (60.4 ± 1.2, 59.2 ± 1.4) and normal apical ridge (82.9 ± 0.5, 80.7 ± 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. Similarly, sperm abnormalities of head (1.20 ± 0.1, 1.20 ± 0.1), mid piece (0.67 ± 0.1, 0.87 ± 0.1) and tail (11.7 ± 0.2, 11.6 ± 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell^® also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell^® may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender.     

Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa

We investigated the use of duck egg yolk (DEY), Guinea fowl egg yolk (GFEY) and Indian indigenous hen (Desi) egg yolk (IDEY) in extender for improving the post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa, and compared it with commercial hen egg yolk (CHEY; control). For this purpose, two consecutive ejaculates of semen from each of two Nili-Ravi buffalo bulls were collected on 1 day each week for 5 weeks (replicates; n=5) with artificial vagina (42 degrees C). Split pooled ejaculates, were diluted in tris-citric acid glycerol extender containing either DEY or GFEY or IDEY or CHEY at 37 degrees C. Extended semen was cooled to 4 degrees C in 2 h and equilibrated for 4 h at 4 degrees C. Cooled semen was then filled in 0.5 ml straws at 4 degrees C and frozen in programmable cell freezer. Thawing of semen was performed at 37 degrees C for 30 s. Sperm motility, plasma membrane integrity and sperm morphology (acrosome integrity, head, mid-piece and tail abnormalities) of each semen sample were assessed at 0, 3 and 6 h after thawing and incubation at 37 degrees C. Visual motility (%) and percentage of intact plasma membranes assessed at 6h post-thaw of buffalo bull spermatozoa were highest (P<0.05) due to DEY as compared to GFEY, IDEY and control. The percentage of spermatozoa with normal acrosomes at 0, 3 and 6 h post-thaw was highest (P<0.05) in DEY extender than GFEY, IDEY and CHEY. Sperm tail abnormalities (%) observed at 0, 3 and 6 h post-thaw in samples cryopreserved with freezing extender having DEY were lower (P<0.05) as compared to extender containing GFEY, IDEY and CHEY. In conclusion, DEY compared to other avian yolks in extender improves the frozen-thawed quality of buffalo bull spermatozoa.     

Spirulina platensis extract addition to semen extender enhances cryotolerance and fertilizing potentials of buffalo bull spermatozoa

The objective of this study was to investigate the effect of Spirulina platensis extract (SPE) addition to the freezing extender on freezability, lipid peroxidation, ultrastructure alterations and fertilizing potentials of frozen-thawed buffalo bull spermatozoa. Semen samples were collected with artificial vagina from five adult fertile bulls and diluted with Tris-base extender containing SPE (1, 5, 10 and 20 μg/mL) or without SPE (control). Diluted semen was cooled to 4 °C throughout one hour and frozen in 0.25 mL straws: prior to being stored in liquid nitrogen. Cryopresreved spermatozoa were assessed for post-thawing sperm motility, viability, acrosomal integrity, ultrastructure changes, antioxidant activities, lipid peroxidation and fertility rate. The current results clearly indicated that adding 10μg/mL SPE to the freezing extender significantly improved (P< 0.05) post-thawing motility and decrease the percentage of acrosomal damage (51.67±6.02% and 16.33±1.46%, respectively) compared with the control (28.33±4.41% and 26.33±1.77%, respectively). Moreover, addition of 10 μg/mL SPE to the semen extender significantly diminished (P< 0.05) MDA concentration (10.66±2.40 nmol/10⁹) compared with the control (22.66±4.26 nmol/10⁹). Therefore, the present results revealed that addition of 10μgl/mL SPE to the freezing extender might improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.     

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.     

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10⁶ spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest (P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.     

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n = 18) were split into five aliquots for dilution (37 °C; 50 × 10⁶ spermatozoa ml^?1) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0 mM GSH. Extended semen was cooled to 4 °C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5 cm above the LN₂ level) for 10 min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4 h post-thawing (37 °C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p < 0.05) in all experimental extenders containing GSH when compared to the control extender (0 mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n = 2) spermatozoa was higher (p < 0.05) in extender containing 2.0 mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0 mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.     

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.     

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).     

Supplementation of l-tryptophan (an aromatic amino acid) in tris citric acid extender enhances post-thaw progressive motility,plasmalemma, mitochondrial membrane potential,acrosome, and DNA integrities,and in vivo fertility rate of buffalo (Bubalus bubalis) bull spermatozoa

The aromatic amino acid l-tryptophan is an essential and versatile molecule, acts by transferring an electron to free radicals and protects the plasma membrane from injuries. The aim of the present study was to investigate the effects of l-tryptophan in extender on semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during cryopreservation. Two ejaculates were collected from each bull (n = 2 ejaculates and n = 4 bulls) with artificial vagina at 42 °C followed by initial evaluation for volume, motility, concentrations and were diluted in five extenders (C = lacking l-tryptophan, D1 = 25 μ M l-tryptophan, D2 = 50 μ M l-tryptophan, D3 = 75 μ M l-tryptophan, and D4 = 100 μ M l-tryptophan) respectively, and cryopreserved. The experiment was repeated four times (n = 4 replicates). At post-dilution, sperm plasma membrane integrity (PMI, %), supravital plasma membrane integrity (SVPMI, %), hypo-resistivity (HR, %) and acrosome integrity (ACR-I, %) were significantly higher (P < 0.05) in extender supplemented with D4 than control. At post-thawing, progressive motility (PM, %), PMI, SVPMI, HR, ACR-I, and DNA-I of buffalo bull spermatozoa were significantly higher in D4 than control. Sperm in vitro longevity (%) assessed in terms of PM, SVPMI, and ACR-1 were significantly higher in D4 than control. Sperm mitochondrial membrane potential (%) was higher in treated groups than the control. The in vivo fertility rate was significantly higher in D4 than control (60.17% vs. 44.17%, P < 0.05). It is concluded that the supplementation of l-tryptophan in tris citric acid extender improves semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during freezing and thawing process.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

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.     

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).     

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.     

Comparison of Biociphos-Plus and TRIS-egg yolk extender for cryopreservation of bull semen

For optimizing routine freezing of bull semen, we examined three different cryopreservation methods using either TRIS-egg yolk-citrate extender or Biociphos-Plus. Biociphos-Plus (IMV, France) has been marketed as an extender, in which egg yolk is replaced by a sterile soybean extract to reduce the contamination risk derived from animal borne substances. We used 78 bulls of various breeds (Brown Swiss, Holstein, Simmental) between 12 and 23 months of age, and we produced a total of 800-1000 straws (0.25 ml, 20 x 10(6) spermatozoa) from each bull using three different methods. In method A, we used TRIS-egg yolk as extender and packaged at 4 degrees C. In method B, we also used TRIS-egg yolk but packaged at room temperature (RT) between 18 and 22 degrees C. In method C, Biociphos-Plus served as extender and we packaged at RT. We compared methods A, B and C by using post-thaw motility, viability, morphology and osmotic resistance as semen quality parameters. In addition, we recorded 75-day nonreturn rates (NR75) to detect the effect of extenders on fertility. With the exception of primary defects, all laboratory parameters investigated were significantly (P < 0.05) better in methods A (TRIS-egg yolk, 4 degrees C) and B (TRIS-egg yolk, RT), compared to method C (Biociphos-Plus, RT). We recorded no significant difference between methods A and B. We could not verify the differing laboratory results by fertility data (NR75). However, when we analyzed NR75 for a single breed, significant (P < 0.05) differences existed between methods A and B compared to method C in Simmental and Holstein but not in Brown Swiss. We obtained best results in Simmental using method A (69%, n = 3384), while method C (61.4%, n = 763) was superior to methods A (57.6%, n = 698) and B (57.3%, n = 737) in Holstein. After considering various factors like preparation of extender, cost of materials and ambient working temperature, we concluded from our data that bull semen processing using TRIS-egg yolk extender and RT for packaging (method B) produced the best semen quality and field fertility.     

Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3 h. Results showed that total motility at 1 and 3 h, progressive motility at 3 h, positive hypoosmotic response at 2 and 3 h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6 h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population.     

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.     

Ram seminal plasma improves pregnancy rates in ewes cervically inseminated with ram semen stored at 5 °C for 24 hours

In this study, we compared pregnancy rates obtained using ram semen stored at 5 °C for 24 h, with ram or bull seminal plasma (SP) added to TRIS-egg yolk extender. During the breeding period, 670 adult Corriedale ewes were cervically inseminated with semen (2 × 10⁸ sperm in a volume of 0.2 mL) from eight adult Corriedale rams. Ejaculates, obtained using an artificial vagina, were split into three aliquots and diluted with the following: TRIS-egg yolk based extender (T), T + 30% ram SP (R), or T + 30% bull SP (B). Samples were refrigerated and stored at 5 °C for 24 h until used for AI. Pregnancy was assessed by ultrasonography 35 to 40 d after AI. Pregnancy rate was not affected by ram (P = 0.77) or breeding period (P = 0.43), and there were no interactions between extender and ram (P = 0.94), or extender and breeding period (P = 0.24). However, there was an effect of extender (P = 0.0009) on pregnancy rates; ram SP, but not bull SP, increased pregnancy rates compared with extender without SP (49.7, 38.1, and 31.1%, for R, B, and T respectively). In conclusion, ram SP added to TRIS-egg yolk extender had a beneficial effect on the pregnancy rate of ram sperm stored at 5 °C for 24 h and used for cervical insemination of ewes.     

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.     

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.