Cryopreservation of the milt of the northern pike

Seven published extenders, three thawing media and two thawing temperatures were tested in order to determine their suitability for cryopreservation of northern pike ( Esox lucius L.) sperm. Sperm motility during successive steps of cryopreservation was evaluated. Erdahl and Graham's extender with the addition of egg yolk proved to be the most efficient (maximum hatching rate of 74.5%) when semen was thawed in 120 m m NaCl solution warmed up to 30° C. No correlations between motility of sperm (diluted in extenders or diluted in extenders and then activated with thawing solution) and the subsequent hatching success were observed. The relationship between motility of thawed sperm and its fertilization ability was considerable, but correlation was not significant. Spermatozoa frozen in some extenders were frequently motile after thawing but they were not able to fertilize the eggs, this resulted in a poor hatching rate. Depending on the extender, the addition of yolk induced either positive or detrimental effects on fertilization success.     

Studies on liquefaction and storage of ejaculated dromedary camel (Camelus dromedarius) semen

The purpose of this study was to evaluate seminal liquefaction and quality of ejaculated camel semen during storage in different extenders at room (23 degrees C) and refrigeration (4 degrees C) temperature. Semen was collected using an artificial vagina and diluted immediately (1:1), using a split-sample technique, in five extenders [(1) Tris-tes egg yolk, (2) Tris-lactose egg yolk, (3) citrate egg yolk, (4) sucrose egg yolk and (5) Tris-fructose egg yolk], while one fraction was kept without an extender to act as control. The semen was transported to the lab at 37 degrees C, in a portable incubator within half an hour, and thereafter liquefaction of semen was monitored every 15 min. After complete liquefaction of the semen it was evaluated for sperm concentration and morphology and then was extended to a final ratio of 1:3. Aliquots of each semen sample were then stored at refrigeration and room temperature. The average volume of an ejaculate was 4.3+/-0.4 mL and it had a very viscous consistency. The average concentration of spermatozoa was 230.4+/-10.7 x 10(6)mL(-1) and the proportion of spermatozoa with protoplasmic droplets averaged 1.02+/-0.2, while 2.7+/-0.6 and 9.7+/-2.9% had mid-piece and tail abnormalities, respectively. All extended semen samples liquefied within 1.5h at 37 degrees C, however, there was slow liquefaction in the sample without an added extender (control). Best liquefaction was observed in Tris-lactose extender followed by Tris-fructose and citrate egg yolk diluents whereas in the other two extenders there was head-to-head agglutination of the spermatozoa. There was no difference in the initial motility of the spermatozoa in extenders 1-5 after its liquefaction, however, after 24 and 48 h of storage a higher proportion of spermatozoa were motile in extenders 1, 2 and 4 (P<0.05) at both the temperatures. There was a gradual decline in viability of the spermatozoa in all extenders at both the temperatures, although, a high portion of the spermatozoa had intact acrosomes throughout the storage period. It may be concluded that dromedary semen, when added to an extender (1:1) immediately after collection, liquefies within 60-90 min at 37 degrees C. It maintains a high proportion of motile and viable spermatozoa that can survive storage up to 48 h in Tris-lactose egg yolk, Tris-tes egg yolk and sucrose egg yolk diluents. However, best liquefaction and progressive sperm motility is achieved in Tris-lactose egg yolk extender.     

Cryopreservation of common carp (Cyprinus carpio L.) sperm I. The importance of oxygen supply

Experiments were carried out on the cryopreservation of common carp ( Cyprinus carpio L.) sperm. The effects of pre-freezing oxygen supply on post-thaw motility and the efficacy of different extenders were studied. Sperm diluents contained DMSO as a cryoprotectant in 10% final concentration. The dilution rate was 1:9 (sperm:diluent). Sperm was diluted and equilibrated (10 min) at 0°C. Sperm was then frozen in plastic straws (0.5 ml) at the following rate: 0°C–4°C: 4°C min⁻¹−4°C–80°C: 11°C min⁻¹ from −80°C, straws were plunged directly into liquid nitrogen (−196°C) for further storage. Frozen samples were thawed in a water bath at 40°C.
The freezability of common carp sperm showing reduced motility (due to suboptimal oxygen supply) after transportation could be restored when 30 min of oxygenation was applied prior to freezing. Highest post-thaw motility (57%, percent of control) was achieved when sperm was diluted with modified Kurokura's 'Extender 2'.     

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 degrees C, and motility assessed after 24h, while the second set was kept at 23 degrees 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 degrees C respectively. At 23 degrees 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 degrees 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 lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa

The effect of lactose and glycerol concentration, as well as the equilibration time with glycerol was studied on motility, normal apical ridge (NAR), and chromatin state of boar spermatozoa after the freezing and thawing process. In the first experiment, samples were frozen in first and second extenders containing different concentrations of lactose (11, 12 and 14%). In the second experiment samples were frozen using second extenders with different concentrations of glycerol (4, 6, 8 and 10%) and were incubated at 5 degrees C for 0 and 30 min. Motility, motility after caffeine treatment, NAR, chromatin condensation and stability (susceptibility to de-condense after heparin treatment) were evaluated. The results indicated that freezing spermatozoa in extenders with increasing concentrations of lactose adversely affected motility but provided a protective effect on acrosomes. Increased lactose concentration induced higher chromatin condensation but maintained the same stability. Increasing the glycerol concentration in the second extender from 4-6 to 8% led to higher motility and NAR as well as lower chromatin condensation and stability. When 30 min equilibration time was allowed after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. The longer equilibration time was detrimental for motility when freezing in the 8% glycerol extender but favourable when using the 4% glycerol extender. Compared to the 8% glycerol, spermatozoa frozen in the 10% glycerol extender showed similar motility and increased chromatin condensation and stability, as well as low values of NAR that did not improve by longer incubation time.     

Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 degrees C. Total motility after 24 h of storage was lowest in semen diluted with PBS (p<0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly (p<0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly (p<0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender (p<0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.     

Effect of solid storage at 15 degrees C on the subsequent motility and fertility of rabbit semen

We conducted two studies to improve preservation of rabbit semen. The objective of the first study was determine whether a glucose- and fructose-based extender with two different amounts of gelatin would solidify at 15 degrees C, and to evaluate the influence of gelatin supplementation on sperm motility parameters after storing semen up to 10 days at 15 degrees C. The fertility of rabbit semen diluted in the best gelatin-supplemented extender established in Study 1 and stored for up to 5 days was evaluated in the second study. In Study 1, semen was collected with an artificial vagina from 40 bucks. Each ejaculate was diluted to (80-100) x 10(6) spermatozoa/mL (1:3, semen/extender) at 37 degrees C in one of the three following glucose- and fructose-based extenders: control (standard liquid extender), semi-gel or gel (0.7 or 1.4 g gelatin in 100 mL extender, respectively). Pools of semen were allocated among 0.6 mL plastic artificial insemination (AI) guns. Thirty (10 per extender group) AI doses were immediately analyzed (0 h) and the remainder stored in a refrigerator (15 degrees C) for 12, 24, 36, 48, 72, 96, or 240 h. All doses with gelatin extenders solidified at 15 degrees C. Semen samples, prewarmed to 37 degrees C, were evaluated with a computer-assisted sperm analysis (CASA) system. The percentage of motile cells was significantly lower using the liquid compared to the gel extenders during semen storage from 0 to 96 h. Although significance was lost, these differences persisted after 240 h of storage. Motility of spermatozoa in the semi-gel extender was intermediate between that of liquid and gel extender throughout the study. Study 2 was performed on 1250 multiparous lactating does. Five homogeneous groups of 250 does previously synchronized were inseminated using semen previously stored for 120, 96, 72, 48 or 24 h, respectively. Rabbit does receiving 24 h-stored semen (diluted with the control extender used in Study 1) served as controls. The remaining females received seminal doses supplemented with 1.4 g/100mL gelatin (gel extender used in Study 1). Kindling rates for rabbit does inseminated with gelatin-supplemented (solid) semen doses stored for 48 h (88%) or 72 h (83%) were similar to those recorded for liquid controls stored for 24 h (81%), whereas rates significantly decreased when the semen was solid and stored for 96 h (64%) or 120 h (60%) before AI. In conclusion, rabbit spermatozoa were effectively stored in the solid state at 15 degrees C, with fertility preserved for up to 5 days. Solid storage of rabbit semen would facilitate commercial distribution.     

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).     

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.     

Effects of a lecithin and catalase containing semen extender and a second dilution with different enhancing buffers on the quality of cold-stored canine spermatozoa

In the present study, a diluent containing 0.8% lecithin (Minitube®, Tiefenbach, G) for the cold storage of canine semen was compared to a Tris-egg yolk extender (TRIS-EY) containing 20% egg yolk. For this purpose, aliquots of 10 mixed ejaculates (main fractions) were either diluted with TRIS-EY or with three lecithin extenders containing 0.8% lecithin with or without catalase and tyrosine. All samples were examined by computer assisted sperm analysis (CASA), chlortetracycline assay (CTC) and flow cytometry, sperm chromatin structure assay (SCSA) and zona pellucida binding assay (ZBA). Samples were then cold stored for 8 d and examinations repeated at days 3 and 8. Measurement in the CASA were repeated daily and prior to measurement, each sample was diluted with each of 4 enhancers with or without acetylcarnitine. The use of an enhancer proved to be essential for all extenders and after 8 d of cooling, progressive motility (P) and viability (V) still averaged > 70% and > 80% with the lecithin extenders containing additives, whereas with TRIS-EY and without additives it was significantly lower (P < 0.05). The percentage of capacitated spermatozoa did not differ between extender groups and there was no significant increase in acrosome reactions (AR) within 3 d. The chromatin status of cells was not changed by cooling within 8 d. The ZBA showed that with additives, significantly more spermatozoa bound to oocytes when a lecithin extender with additives was used (P < 0.05). In conclusion, the 0.8% lecithin extender containing catalase, conserved P and V during 8 d of cold storage better than the TRIS-EY extender, however, only when an enhancer was used; addition of acetylcarnitine to the enhancer did not further improve semen quality. The here introduced lecithin extender / enhancer combination is a useful tool for prolonged storage of cooled semen with excellent longevity and binding ability; addition of tyrosine to the extender did not improve semen quality.     

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 degrees 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 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees 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 degrees 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 egg yolk and glycerol levels in lactose-EDTA-egg yolk extender and of freezing rate on the motility of frozen-thawed stallion spermatozoa

Two experiments were designed to evaluate the effects of egg yolk and glycerol concentrations, freezing rate, and clarification of a lactose-EDTA-egg yolk extender on the post-thaw motility of stallion spermatozoa. In both experiments there was no influence of freezing rate (vapor vs controlled) on the percentage of progressively motile spermatozoa after thawing. Furthermore, no significant interaction among treatments was detected. In Experiment 1, clarified (centrifuged at 34,400 × g for 30 min) lactose-EDTA-egg yolk extenders containing 16 or 20% egg yolk and 3 or 4% glycerol were superior to those containing 12% egg yolk or 2% glycerol, based on the percentage of progressively motile stallion spermatozoa at 0, 30, 60, and 90 min after thawing. However, in Experiment 2, clarification of the lactose-EDTA-egg yolk extender was detrimental to the ability of the stallion spermatozoa to survive after thawing; 4% glycerol was superior to 2% glycerol. The best extender based on the percentage of progressively motile spermatozoa after thawing was nonclarified lactose-EDTA-egg yolk extender containing 20% egg yolk and 4% glycerol.     

Comparison of different extenders for the cryopreservation of Atlantic salmon spermatozoa

Fifteen extenders were produced by adding dimethyl sulfoxide (DMSO) at 8, 10 or 12% of diluent volume to 5 diluents. All extenders were cooled to 4 degrees C. Pooled Atlantic salmon (Salmo salar ) semen with greater than 90% progressive motility was kept at 4 degrees C and added to each extender so that the semen was diluted 1:3 (semen:extender). The equilibration time was less than 5 minutes at 4 degrees C. The extended semen was loaded into 0.5-ml straws and was cooled from 4 degrees C to -90 degrees C at a rate of 30 degrees C per minute. The straws were then plunged into liquid nitrogen for storage. Fluorometry was used to determine the viability of the semen in each of the extenders after freezing and thawing. Cryopreservation of Atlantic salmon semen in Extender 3 (0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4).7H(2)O, 7.5 g/l L-alpha-lecithin and 12% dimethyl sulfoxide) and Extender 12 (0.100 M KHCO(3), 0.0065 M reduced glutathione, 0.125 M sucrose and 12% dimethyl sulfoxide) resulted in significantly (P<0.05) lower percentages of dead spermatozoa than for the other extenders. Furthermore, there was a significantly (P<0.05) lower percentage of dead cells in Extender 3 than in Extender 12.     

Mink semen studies: I. Liquid preservation and prospect of freezing spermatozoa

Using 22 males, 41 semen samples were collected from the vagina of mink by means of a plastic tubing attached to a 1 ml syringe. Subsamples of vaginal semen were diluted in 4 different extenders, viz., tris (tris, citric acid, glycine, fructose, glycerol and egg yolk), PVP (tris extender with polyvinyl pyrrolidone and caproic acid), milk (boiled and filtered milk with glycerol) and sodium citrate. The extended semen samples were stored at 23, 5 and −196°C for varying periods and evaluated for % motile spermatozoa. In the tris extender storage for 3 days at 5°C or for 2 days at 23°C reduced the number of spermatozoa by more than 50%. When milk was used as the extender, the motility decreased from an initial value of 68% to 10% after 5 days of 5°C and to 8% after 4 days at 23°C. The PVP extender was not suitable for storage at any temperature. After being frozen at −196°C for 2 hr, the motility ranged from 3–10% in the tris extender and was zero in milk and PVP extenders. Prolonged storage for 7 days in tris extender reduced the motility to 1–7%.     

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.     

Effect of storage conditions on the LDL effectiveness in ovine sperm cryopreservation

The present study evaluated the effect of storage conditions on the LDL efficacy for cryopreserving ovine sperm. In this way, we compared egg yolk extender with three different forms for LDL storing, LDL diluted in Tris-glucose extender and stored in frozen (i) and freeze-dried (ii) states and LDL stored pure and added into the extender prior to use (iii). We also tested the effect of two storage temperatures (−20 and −80 °C) and three storage times (30, 60, 120 d). Frozen and freeze-dried extenders containing LDL, as well as LDL stored pure, improved post-thaw sperm quality. Storage temperatures did not influence negatively the cryoprotectiveness of LDL extenders. Furthermore, lyophilised LDL extenders stored at −20 °C were more effective in preserving sperm longevity than the other extenders stored at −20 °C. Finally, LDL extenders stored for 30 and 120 d were more efficient than 60 d in preserving ram sperm freezability.     

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me₂SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mg ml⁻¹ propolis) and the group V (1 mg ml⁻¹ propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.     

Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen.Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed.These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.     

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.     

Proteolytic enzyme treatment: an improved method for elimination of egg stickiness in tench, Tinca tinca L., in aquaculture

The aim of the study was to improve the elimination of egg stickiness in tench as an attempt to increase the egg hatching rate. The usual desticking procedure involving treatment with milk solution/clay suspension resulted in a hatching rate of 74.1%. The highest hatching rate of 87.1% was found with a 10.0 ml L^?1 enzyme treatment; hatching rates of 85.2 and 85.1% were recorded at 15.0 and 5.0 ml L^?1, but the hatching rate decreased to 80.0% at 20.0 ml L^?1. anova showed a significant effect of differing concentrations of enzyme and milk solution/clay suspension control (P < 0.0015, P < 0.0003) on the hatching rate and duration of egg incubation until hatching, respectively. Application of proteolytic alcalase enzyme, Merck EC 3.4.21.14, for desticking of tench eggs also decreased the time of egg handling from almost 1 h, necessary for traditional milk/clay desticking, to just 2 min.