Effect Of Different Extenders Research Articles

Effects of extenders and cryoprotectant combinations on motility and morphometry of sea bass (Dicentrarchus labrax) spermatozoa

Summary The aim of this study was to evaluate the effects of different extenders on sea bass (Dicentrarchus labrax) spermatozoa motility and morphology. Six sperm extenders based on inactivator media, DI1 (here named SAN) and Non-Activating Medium (NAM) were tested with European sea bass spermatozoa. The best results were obtained with NAM medium (59.83 mm NaCl, 12.91 mm MgCl2, 1.47 mm KCl, 3.51 mm CaCl2, 20 mm NaHCO3, 0.44 mm glucose) plus 1 and 2% of BSA (NAM1 and NAM2, respectively). The motility of the spermatozoa incubated in those media was similar to the fresh sperm until 48 h (NAM1: 74.3 ± 5.4; NAM2: 78.8 ± 5.8%, and higher than undiluted sperm, 19.1 ± 7.8). We also checked the spermatozoa motility and morphology reactions with some of the best extenders, NAM2 and SAN, and combined them with different concentrations (2, 5, 10%) of three cryoprotectants: methanol, glycerol and dimethyl sulphoxide (DMSO). Glycerol + SAN or NAM2 caused activation of spermatozoa motility, which was lost 5 min later. Methanol and DMSO plus NAM2 extenders resulted in a low activation level and high motility 5 min after incubation, identifying these combinations as good candidates to be used in the cryopreservation of the European sea bass spermatozoa.

The role of the actin cytoskeleton in equine spermatozoal response to osmotic stress

Inseminations with frozen–thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 °C for 24 h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio®, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio® than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen–thawed epididymal sperm showing that Botu-Crio® was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp + Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio® was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.

Freezing of stallion epididymal sperm

Inseminations with frozen–thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 °C for 24 h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio ®, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio ® than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen–thawed epididymal sperm showing that Botu-Crio ® was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp + Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio ® was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.

Effects of glycerol additions on post-thaw fertility of frozen rainbow trout sperm, with an emphasis on interaction between extender and cryoprotectant

Summary The study was conducted to evaluate the effects of different extenders, cryoprotectants and glycerol additions on the post-thaw fertility and interactions between extenders and cryoprotectants during cryopreservation. Semen was collected by abdominal stripping from 30 adult male rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and diluted with three different extenders (Erdahl–Graham, Lahnsteiner, glucose-based) containing 15% DMA, 15% DMSO, 15% DMA + 1% glycerol and 15% DMSO + 1% glycerol at a ratio of 1 : 2. Diluted samples were frozen as 0.1 ml pellets directly on dry ice (solid CO2, −79°C). Eggs were pooled from 10 females. Fertilization was applied in plastic dishes and 600 eggs were used in each fertilization trial. Pellets were thawed in their own extenders (30°C) at a ratio of 1 : 10. 0.3% NaCl was used for activating motility. Sperm–egg ratio was approximately 3 × 106 sp per egg. Experimental success was determined as the percentage of eyed-eggs 25 days after fertilization. The highest eyed-egg rate (49.3%) was obtained from semen frozen with glucose-based extender containing 15% DMSO + 1% glycerol. Our results indicate that the glucose-based extender containing DMSO is a useful combination, but that the addition of glycerol does not have a positive effect on post-thaw fertility, and that interaction of the extender-cryoprotectant is also important in the cryopreservation of rainbow trout semen.

215 EPIDIDYMAL SPERM CRYOPRESERVATION OF ONE SOMALIA WILD ASS (EQUUS AFRICANUS SOMALIENSIS) USING SIX DIFFERENT EXTENDERS

The Somalia wild ass (Equus africanus somaliensis) is a critically endangered taxon (IUCN 2004 red list) which could benefit from biological resource banking. In this work, we studied the effect of different extenders applied to the cryopreservation of epididymal sperm obtained from one male of this subspecies. This animal (13 years old; housed in Cabarceno Park, Cantabria, Spain) was castrated because of very aggressive behavior with other mature males. Genitalia were dissected and weighed (testicles: right, 166 g, and left, 179 g; cauda epididymis: right, 9.3 g, and left, 11.8 g). Sperm were flushed from the cauda epididymis, yielding 15 mL of sample. Sperm concentration was 15 × 109 spermatozoa/mL, totaling 225 × 109 (allowing 4500 doses at 50 × 106 sperm/dose). Sperm motility (TM = % total motile; PM = % progressive; VAP = average path velocity) was assessed by CASA (Microptic, Barcelona, Spain). Viability (VIAB = % viable sperm) and acrosomal status (ACR = % viable spermatozoa with intact acrosomes) were assessed using propidium iodide (37 μmol/L) and PNA-FITC (1 ng/L) and flow cytometry. Chemicals were purchased from Sigma (Madrid, Spain). Part of the sample was divided into six aliquots and diluted 1:1 with different extenders: UL4: Tes-Tris-Fructose (TTF), 10% egg yolk (EG), and 4% glycerol (G); UL8: TTF, 20% EG, and 8% G; AND4: Andromed® (Minitüb, Tiefenbach, Germany) and 4% G; AND7: Andromed® and 7% G; GENT: Gent 1045; and INRA: INRA96 and 4% G. Andromed, Gent, and INRA are commercial extenders. Samples were cooled to 5°C (−0.2°C/min) and then diluted to 200 × 106 sperm/mL. Samples were packed (0.5-mL straws) and frozen using a biofreezer (from 5°C to −15°C at −15°C/min, and from −15°C to −100°C at −25°C/min). Samples were thawed at 65°C for 6 s, and assessed as for pre-freezing (Table 1). Post-thawing motility recovery using AND7 was excellent. The highest viability recovery was achieved by UL4, although that in AND7 was similar. The poor results of equine commercial extender Gent 1045 in this species are remarkable. Our results highlight the importance of species differences in the field of sperm cryopreservation. It is necessary to carry out continuous research for optimizing cryopreservation protocols in order to create germplasm banks for wild species. Table 1. Quality assessment results

Effect of different extenders on sperm viability of buck semen stored at room temperature

The use of egg yolk containing extenders in buck semen requires the washing of the semen due to the presence of egg yolk coagulating enzyme (EYCE) in the seminal plasma of the goat. Egg yolk prevents the effects of cold shock when semen is cooled. Alternatively, storage of buck semen at temperatures above 15 °C allows use of extenders without egg yolk. The aim of the study was to evaluate the effect of different extenders without egg yolk on the viability of buck semen stored at room temperature. Ejaculates were collected once from four adult bucks and split into five aliquots each and diluted with one of five extenders: Skim milk (Mi), Laiciphos ® (Lai), Beltsville Thawing Solution (BTS), and Biociphos Plus ® (Bio-5 and Bio-20). The diluted semen was stored at room temperature, except for Bio-5, which was cooled and stored at 5 °C. Sperm motility and acrosomal status were evaluated following 3, 8, 12, 24 and 28 h. Motility and acrosomal status were significantly influenced by the extenders and storage time ( P < 0.001), while only sperm motility was influenced by the interaction between extender and storage time ( P < 0.001). Progressive motility and acrosome integrity were maintained longer in milk-based extenders, e.g. Laiciphos ®, compared to Biociphos Plus ®. Storage at 5 °C in Biociphos Plus ® was better for sperm viability than storage at 20 °C. No motile spermatozoa were recorded after 3 h of storage in BTS. In conclusion, the results of the trial indicate that milk-based extenders, especially Laiciphos ®, preserved sperm motility and acrosome integrity better than Biociphos Plus ®, when buck semen was stored at 20 °C for up to 28 h.

Comparison of different extenders on the quality characteristics of turkey semen during storage

RiassuntoConfronto di differenti diluenti sulle caratteristiche qualitative del seme conservato di tacchino. È stato valutato l’effetto di tre differenti diluenti: BPSE, Lake e IGGKPh sulla qualità del seme di tacchino durante la conservazione per 48 h a 5°C. Ogni pool di seme è stato diviso in 3 aliquote le quali sono state diluite rispettivamente con i tre medium e determinate in vitro la motilità (procedura Accudenz®), la vitalità (SyBr-PI) e l’integrità di membrana previo stress ipoosmotico. La qualità del seme di tacchino peggiora (P<0,01) durante la conservazione per 48 h, tuttavia i valori di vitalità, mobilità ed integrità di membrana risultano più alti (P<0,01) dopo 24 h e 48 h con BPSE rispetto ai diluenti IGGKPh e Lake.

The effect of different extenders on post-thaw sperm survival, acrosomal integrity and longevity in cryopreserved semen of Formosan Sika deer and Formosan Sambar deer

This study investigates the efficacy of five extenders in contributing to the outcome of semen cryopreservation in Formosan Sika and Sambar deer. Pooled semen ( n=4) of six males of each breed was used. In Sika deer, semen collection rate was 96% (23/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 0.5±0.4 ml, 77±6% and 1471.3±940.0×10 6 ml −1, respectively. Post-thaw motility in respective extender was A: 66±16%; B: 71±2%; C: 73±6%; D: 9±4% and E: 26±12% (mean±S.D.). In extender C (74±14%) more viable spermatozoa were preserved than in the others (A: 64±10%; B: 48±11%; D: 41±16%; E: 47±6%; P<0.05). Acrosomal integrity was not influenced by extender composition. Post-thaw motility did not decrease during a 4-h incubation period, irrespective of the extender used ( P>0.05). In Sambar deer, semen collection rate was 88% (21/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 1.3±0.5 ml, 82±4% and 379.1±252.2×10 6 ml −1, respectively. Post-thaw motility was in respective extenders A: 69±2%; B: 74±6%; C: 73±2%; D: 13±6% and E: 31±20%. Extenders B and C were superior ( P>0.05) with respect to sperm motility. Similarly, post-thaw viability in extenders A (70±7%), B (76±7%) and C (79±2%) was higher than that D (25±19%) and E (29±17%) ( P<0.01). Sperm acrosomal integrity was better preserved in extenders B (86±4%) and C (83±4%) than in extenders A (54±13%), D (39±22%) and E (46±22%) ( P<0.05). Post-thaw sperm longevity in extender A reduced from 69 to 16% during incubation ( P<0.05) whereas only a slight decrease was observed in the other extenders after 4 h. In conclusion these data show that egg-yolk–Tris–Tes–glycerol based extender C containing Equex STM paste is optimal for freezing semen of Formosan Sika deer while egg-yolk–Tris–citric acid–glycerol based extender B containing Equex and extender C are superior in semen cryopreservation to others for Formosan Sambar deer.

Preliminary study on the cryopreservation of tropical bagrid catfish ( Mystus nemurus) spermatozoa; the effect of extender and cryoprotectant on the motility after short-term storage

The effects of different extenders, and cryoprotectants on the motility of tropical bagrid catfish ( Mystus nemurus) spermatozoa were evaluated after short-term storage. Three extenders, physiological saline, Ringer or saline at three levels of sperm to extender dilutions (1:20, 1:30, or 1:40) and four cryoprotectants (DMSO, ethanol, glycerol or methanol) at three concentrations (5, 10, or 15%) were examined in two separate experiments. In the first experiment, milt was suspended in the respective extender at the three milt to extender dilution ratios in two sets of tubes. Extended milt in the first set of tubes was stored at −4 °C, and motility assessed after 24 h, while the second set was kept at 23 °C and sperm motility was assessed immediately and at 30-min intervals thereafter. Ringer retained sperm motility better than the other extenders at all dilution levels at temperatures of 23 and −4 °C respectively. At 23 °C, the sperm motility was almost completely lost after 150 min except for those in Ringer at 1:20 dilution level which still had a motility of 18% (compared to those kept at −4 °C for 24, which had motility from 39 to 71%, regardless of extender). In the second experiment, various cryoprotectants were added to solutions of milt (that was diluted in Ringer at 1:20 ratio and cryopreserved in liquid nitrogen for 15 days). Sperm cryopreserved in 10% methanol had the highest motility (58%) compared with those in the other cryoprotectants at all concentrations.

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 °C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac ® and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 °C seemed to influence sperm viability parameters less than storage at 20 °C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 °C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.

Effects of different extenders and holding temperatures on viability of spermatozoa from the scimitar-horned oryx ( Oryx dammah)

Effects of different extenders and holding temperatures on viability of spermatozoa from the scimitar-horned oryx ( Oryx dammah)

Effect of different extenders on glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) release from frozen buffalo semen

Sixty buffalo semen samples (motility greater than 60%) were frozen in 3 extenders, viz., Tris yolk glycerol (TY-G), Citric acid whey glycerol (CAW-G) and Egg yolk glucose sodium bicarbonate glycerol (EYGSB-G) for studying the release of GOT and GPT enzymes in the extracellular fluid during pre-freezing (after first extension) and post-freezing (15 minutes and 30 days after freezing). Release of GOT and GPT enzymes was less in TY-G than CAW-G and EYGSB-G extenders. Significant differences (P<0.01) in GOT and GPT release were observed between extenders and bulls at various stages of freezing of semen.