Effect of seminal extenders containing egg yolk and glycerol on motion characteristics and fertility of stallion spermatozoa

Three experiments were conducted to evaluate the effects of egg yolk and(or) glycerol added to a nonfat dried skim milk-glucose (NDSMG) extender on motion characteristics and fertility of stallion spermatozoa. In Experiment 1, ejaculates from each of 8 stallions were exposed to each of 4 extender treatments: 1) NDSMG, 2) NDSMG + 4% egg yolk (EY), 3) NDSMG + 4% glycerol (GL), and 4) NDSMG + 4% egg yolk + 4% glycerol (EY + GL). Samples were cooled at -0.7 degrees C/min from 37 to 20 degrees C; subsamples were then cooled at -0.05 or -0.5 degrees C/min from 20 to 5 degrees C. Percentages of motile spermatozoa (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, 24 and 48 h after initiation of cooling. There was no overall effect (P > 0.05) of cooling rate. PMOT was highest (P < 0.05) for spermatozoa extended in NDSMG + GL at 48 h. At 24 and 48 h, MOT and PMOT were lowest (P < 0.05) for spermatozoa extended in NDSMG + EY. In Experiment 2, ejaculates from 8 stallions were exposed to each of 4 treatments: 1) NDSMG, 2) NDSMG + EY, 3) semen centrifuged in NDSMG and resuspended in NDSMG, and 4) semen centrifuged in NDSMG and resuspended in NDSMG + EY. Samples were cooled from 20 to 5 degrees C at each of 2 rates (-0.05, -0.5 degrees C/min). A detrimental interaction between seminal plasma and egg yolk was noted for PMOT at 6 h and for both MOT and PMOT at > or = 24 h postcooling. Experiment 3 determined if egg yolk or glycerol affected fertility. The seminal treatments were 1) NDSMG, 2) NDSMG + EY with previous removal of seminal plasma, and 3) NDSMG + GL. All samples were cooled to 5 degrees C and stored 24 h before insemination. Embryo recovery rates 7 d after ovulation were lower for mares inseminated with spermatozoa cooled in NDSMG + EY (17%, 4/24) or NDSMG + GL (13%, 3/24) extenders, than semen cooled in NDSMG (50%, 12/24). We concluded that egg yolk (with seminal plasma removal) or glycerol added to NDSMG extender did not depress MOT or PMOT of cooled stallion spermatozoa but adversely affected fertility.     

Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at -20 degrees C until warmed to 37 degrees C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated (r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of egg yolk, glycerol and cooling rate

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.     

Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa

Four experiments were conducted to investigate the effects of egg yolk during the freezing step of cryopreservation (namely, the process except for the cooling step), on the viability of goat spermatozoa. The effects of egg yolk on sperm motility and acrosome integrity during the freezing step were investigated in Experiment 1. Spermatozoa diluted with Tris-citric acid-glucose (TCG) solution containing 20% (v/v) egg yolk were cooled to 5 degrees C, washed, and then frozen in TCG with egg yolk (TCG-Y), TCG without egg yolk (TGG-NY), 0.370 M trehalose with egg yolk (TH-Y), or trehalose without egg yolk (TH-NY). All extenders contained glycerol. In frozen-thawed spermatozoa, the inclusion of egg yolk in the freezing extenders increased (P<0.05) percentages of motile sperm, progressively motile sperm, and the recovery rate (ratio of post-thaw to pre-freeze values), but decreased (P<0.05) acrosomal integrity. Moreover, extenders with trehalose had better (P<0.05) post-thaw sperm viability. In Experiment 2, the effects of egg yolk on acrosome status before and after freezing were studied. Egg yolk significantly decreased the proportion of intact acrosomes before freezing, leading to fewer (P<0.05) intact acrosomes post-thaw and lower (P<0.05) recovery rates for intact acrosomes. In Experiment 3, including sodium dodecyl sulfate (SDS) in a diluent containing egg yolk tended to preserve the acrosome compared with the egg yolk containing diluent free of SDS, however, spermatozoa had a lower (P<0.05) proportion of intact acrosomes than those in a yolk-free diluent. However, after cooling, spermatozoa were diluted with a glycerolated extender containing egg yolk. Therefore, the objective of Experiment 4 was to explore whether the egg yolk or glycerol was responsible for the reduced intact acrosome percentage. In this experiment, after cooling and washing the spermatozoa were diluted in TCG with glycerol and/or egg yolk. The combination of glycerol and egg yolk in the extender reduced (P<0.05) the proportion of intact acrosomes compared with egg yolk or glycerol alone. In conclusion, the inclusion of egg yolk significantly improved sperm motility, indicating its beneficial effects during the freezing step of cryopreservation; trehalose appeared to synergistically increase its cryoprotective effects. Furthermore, although neither glycerol nor egg yolk per se affected the proportion of intact acrosomes, the combination of the two significantly reduced the proportion of acrosome-intact spermatozoa.     

Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa

The effects of the dialyzable fractions from bovine seminal plasma, egg yolk, and milk and of two buffer systems (TEST and sodium citrate) on post-thaw sperm motility were studied. Each basic salt solution was used in the experimental design. These solutions were used as extender systems in combination with egg yolk and glycerol. After collection, semen samples were extended (1:20), cooled to 5 degrees C in 1.5 hr, and frozen in 0.5-cc French straws after 3 hr of equilibration. Post-thaw samples were assayed for percentage of motile cells immediately after thawing and after 4 hr of incubation at room temperature (22 degrees C). Egg yolk (25%) provided the same protection as did the combination of colloidal material present in the skim milk-yolk extenders. The use of TEST as a buffer provided significantly higher (P less than 0.01) sperm post-thaw motility than milk salts or Na citrate. Sperm survival in extenders containing high concentrations of seminal plasma and/or egg yolk salts was significantly lower (P less than 0.01). Spermatozoa frozen in the presence of 6% glycerol resulted in sperm motility significantly (P less than 0.05) higher than that of spermatozoa frozen with 3% glycerol. However, no difference was observed between these two concentrations when TEST solution was used.     

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.     

Effect of cooling of equine spermatozoa before freezing on post-thaw motility: preliminary results

The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5 degreesC and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P < 0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P < 0.05). The percentages of PM spermatozoa frozen in 0.5- or 2.5-mL straws were similar (21 and 28%, respectively; P > 0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 degreesC) or after cooling (5 degreesC), and spermatozoa were frozen after being cooled to 5 degreesC for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P < 0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25 degreesC then cooled for 12 h to 5 degreesC had higher (P < 0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5 degreesC (13%). In the third experiment, spermatozoa were centrifuged for seminal plasma removal, resuspended at spermatozoal concentrations of 50,250 or 500 x 10(6)/mL, cooled to 5 degreesC for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10(6)/mL had higher (P < 0.05 percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10(6) spermatozoa/mL (17%). We recommend that semen be centrifuged at 25 degreesC to remove seminal plasma, suspended to 250 x 10(6) spermatozoa/ml and held at 5 degreesC for 12 h prior to freezing.     

Insemination of mares with low numbers of either unsexed or sexed spermatozoa

Two experiments were conducted to determine pregnancy rates in mares inseminated 1) with 5, 25 and 500 x 10(6) progressively motile spermatozoa (pms), or 2) with 25 x 10(6) sex-sorted cells. In Experiment 1, mares were assigned to 1 of 3 treatments: Group 1 (n=20) was inseminated into the uterine body with 500 x 10(6) pms. Group 2 (n=21) and Group 3 (n=20) were inseminated into the tip of the uterine horn ipsilateral to the preovulatory follicle with 25 and 5 x 10(6) pms, respectively. Mares in all 3 groups were inseminated either 40 (n=32) or 34 h (n=29) after GnRH administration. More mares became pregnant when inseminated with 500 x 10(6) (18/20 = 90%) than with 25 x 10(6) pms (12/21 = 57%; P<0.05), but pregnancy rates were similar for mares inseminated with 25 x 10(6) vs 5 x 10(6) pms (7/20 = 35%) (P>0.1). In Experiment 2, mares were assigned to 1 of 2 treatments: Group A (n=11) was inseminated with 25 x 10(6) spermatozoa sorted into X and Y chromosome-bearing populations in a skimmilk extender. Group B (n=10) mares were inseminated similarly except that spermatozoa were sorted into the skimmilk extender + 4% egg yolk. Inseminations were performed 34 h after GnRH administration. Freshly collected semen was incubated in 224 microM Hoechst 33342 at 400 x 10(6) sperm/mL in HBGM-3 for 1 hr at 35 degrees C and then diluted to 100 x 10(6) sperm/mL for sorting. Sperm were sorted by sex using flow cytometer/cell sorters. Spermatozoa were collected at approximately 900 cells/sec into either the extender alone (Group A) or extender + 4% egg yolk (Group B), centrifuged and suspended to 25 x 10 sperm/mL and immediately inseminated. Pregnancy rates were similar (P>0.1) between the sperm treatments (extender alone = 13/10, 30% vs 4% EY + extender = 5/10, 50%). Based on ultrasonography, fetal sex at 60 to 70 d correlated perfectly with the sex of the sperm inseminated, demonstrating that foals of predetermined sex can be obtained following nonsurgical insemination with sexed spermatozoa.     

Epididymal and ejaculated cat spermatozoa are resistant to cold shock but egg yolk promotes sperm longevity during cold storage at 4 degrees C

The aims were to evaluate the susceptibility of feline ejaculated and epididymal spermatozoa to cold shock and to evaluate the effect of egg yolk in the preservation extender. Ejaculated and epididymal spermatozoa from eight males were subjected to a slow (0.5 degrees C/min) or a fast (3 degrees C/min) cooling rate with controls kept in room temperature. Ejaculated and epididymal spermatozoa from another eight males were cooled in a plain Tris buffer (Tris) or in Tris with 20% egg yolk (EYT) and evaluated for 96 h. Subjective motility (MOT), plasma membrane integrity (PMI), and acrosome integrity (ACRI) were evaluated. Cooling did not induce sperm damage regarding PMI (P=0.6) or ACRI (P=0.19) and chilled spermatozoa had better overall MOT (P=0.046) than controls. EYT was better for MOT (P>0.05) from 48 h of cold storage than Tris. EYT was also better for overall ACRI (P<0.0001) while Tris was better for overall PMI (P=0.0004). There were no interactions between time and treatment (P>0.05) for PMI or ACRI. Ejaculated spermatozoa had better overall MOT (P<0.05) and PMI (P<0.05) than epididymal spermatozoa, and higher ACRI in experiment 1 (P=0.0003) but not in experiment 2 (P=0.117). Source of spermatozoa did not affect the susceptibility to cooling or the effect of egg yolk as there were no interactions (P>0.05) between source of spermatozoa and treatment (cooling or control) or between time, source and extender (P>0.05). In conclusion cat spermatozoa were tolerant to cold shock and egg yolk was beneficial for preservation of MOT and ACRI but not PMI.     

Microscopic and flow cytometric semen assessment of Dutch AI-bucks: effect of semen processing procedures and their correlation to fertility

This study was done to determine the effects of processing techniques on the quality of semen from Dutch AI-bucks with the view on improving pregnancy rates after artificial insemination (AI) with liquid or frozen-thawed semen. Motility of spermatozoa was estimated under a microscope whereas the percentage live spermatozoa and the percentage live spermatozoa with intact acrosomes were determined by means of flow cytometry. Aspects of semen processing that were investigated are storage temperature of liquid semen (i), the effect of glycerol on liquid-stored semen (ii), removal of seminal plasma (iii) and type of extender (iv). The correlation between semen quality and fertility rates in inseminated does was also investigated. The percentage motile spermatozoa in semen stored in liquid form for 72 h progressively declined over time, irrespective of whether storage occurred at 4 or 18 degrees C. The percentage motile spermatozoa in semen stored at 18 degrees C was similar to that in semen stored at 4 degrees C if stored for 24 h but lower if stored for 48 h. Goats differ in the sensitivity of their spermatozoa to the deleterious effects of glycerol. Neither the removal of seminal plasma nor the type of extender had any effect on semen quality before freezing but semen frozen in a Tris-citric acid-glucose (TCG) buffer with egg yolk without removal of the seminal plasma had better quality after thawing than semen frozen in another diluent or after removal of seminal plasma. Remarkably no significant correlation between fertility and membrane integrity of spermatozoa could be found. Thus, although integrity assays for spermatozoa are useful to asses resistance to semen handling, the validity of these assays for predicting fertility is questioned.     

Osmotic characteristics of mouse spermatozoa in the presence of extenders and sugars

Successful cryopreservation requires cells to tolerate volume excursions experienced during permeating cryoprotectant equilibration and during cooling and warming. However, prior studies have demonstrated that mouse spermatozoa are extremely sensitive to osmotically induced volume changes. A series of three experiments were conducted 1) to test the efficacy of two commonly used extender media components, egg yolk (EY) and skim milk (SM), in broadening the osmotic tolerance limits (OTL) of ICR and B6C3F1 murine spermatozoa; 2) to determine if the extender components affected sperm plasma membrane permeability coefficients for water and cryoprotective agent (CPA) characteristics; and 3) to test the effects of permeating and nonpermeating CPA on mouse sperm morphology. In experiment 1, sperm samples were added to 150, 225, 300, 450, or 600 mOsm NaCl, EY, SM, sucrose, or choline chloride at 22 degrees C and then returned to isosmotic conditions. In experiment 2, epididymal sperm were preequilibrated in 1 M glycerol (Gly) or 2 M ethylene glycol (EG) prepared in SM extender, abruptly exposed to isosmotic conditions at 22, 15, or 2 degrees C, and the corresponding volume excursions were measured and analyzed. In experiment 3, the effects of permeating CPA (0.3 M EG or dimethyl sulfoxide) or nonpermeating CPA (12% sucrose or 18% raffinose) on sperm morphology (i.e., principle midpiece folding and putative membrane fusion) were evaluated. Experiment 1 showed that spermatozoa from ICR and B6C3F1 mice have effectively broader OTL when exposed to EY or SM extenders. The results of experiment 2 indicated that, for ICR sperm, the activation energy (E(a)) for the hydraulic conductivity (L(p)) was unchanged in SM extender. However, for B6C3F1 sperm, there were significant differences in E(a) of L(p) in the presence of Gly and EG. The result of experiment 3 indicated that permeating CPAs damage sperm membrane integrity, causing a high frequency of head-to-tail or tail-to-tail membrane fusion, whereas this occurrence in the presence of nonpermeating CPA was less than 3%. Finally, the results of experiments 1 and 2 were combined in a mathematical model to predict Gly and EG addition and removal in the presence of SM extender, which would prevent mouse sperm membrane damage. These predictions indicated that, for ICR sperm, both Gly and EG may be added and removed in a single step. However, for B6C3F1 spermatozoa, Gly required a two-step addition while EG only required a single step. For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal.     

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.     

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.     

Effect of glycerolization procedure and removal of seminal plasma on post-thaw survival and got-release from Boer goat spermatozoa

Forty ejaculates (20 for each of 2 experiments) were collected from 4 Boer goat bucks at weekly intervals to study the effect of glycerolization procedure and removal of seminal plasma on progressive motility, percent live spermatozoa and release of glutamic oxaloacetic transaminase (GOT) before and after the freezing of semen. Stepwise glycerolization at 37 degrees C gave higher progressive motility and percentage of live spermatozoa both before freezing and after thawing than onestep glyceroliza-tion at 37 degrees C or stepwise extension with glycerol being added after cooling to 5 degrees C. The GOT-release was reduced before freezing and after thawing of semen with stepwise glycerolization (P < 0.05). Progressive motility and the percentage of live spermatozoa were higher (P < 0.05) after the freezing of whole semen than in washed spermatozoa. The concentration of GOT in the extra-cellular fluid was lower in washed spermatozoa prior to freezing (P < 0,05); but after thawing, the washed spermatozoa released more GOT than spermatozoa in whole semen. Removal of seminal plasma prior to freezing spermatozoa in an extender containing egg yolk had an unfavorable effect on their post-thaw motility and integrity.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Influence of different semen extenders and seminal plasma on PMN migration and on expression of IL-1beta, IL-6, TNF-alpha and COX-2 mRNA in the equine endometrium

After artificial insemination or mating an inflammatory response is induced by spermatozoa and components of the inseminate or ejaculate. In order to investigate the inflammatory reaction of the endometrium to different semen extenders, phosphate buffered saline (PBS), seminal plasma (SP), skim milk-based extender (SM) or egg yolk semen extender (EY) was inoculated into the uterus of oestrous mares (n=8) during four consecutive cycles in alternating order. Twelve hours after treatment, a uterine lavage was performed and an endometrial biopsy was taken. An additional biopsy was taken in the oestrous cycle before experiments were started. No differences in volume, pH, specific density or cell count of lavage fluid were found between the treatments. A significantly (p<0.01) lower number of leukocytes in the endometrium was identified in pre-experiment biopsies (68+/-5 leukocytes per field) compared to PBS (154+/-32), SP (175+/-22), SM (193+/-29) and EY treatments (113+/-17). PMN numbers were lower (p<0.01) after infusion of EY (23+/-10) compared to PBS (59+/-21) and SM extender (69+/-21). The number of eosinophils increased after inoculation of SP (p<0.05 vs. PBS, SM and EY). All treatments increased expression of interleukins (IL)-1beta and 6, tumor necrosis factor-alpha (TNF-alpha) and cyclooxgygenase-2 (COX-2) in the endometrium compared to pre-experiment values. Expression of COX-2 mRNA was significantly higher after infusion of SM than after PBS treatment (p<0.04). In conclusion, extender alone as well as seminal plasma and PBS causes an inflammatory endometrial response with the least pronounced response induced by EY-based semen extender.     

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.     

Fertilizing potential of mouse spermatozoa cryopreserved in a medium containing whole eggs

Experiments were conducted to improve survival of mouse spermatozoa through the cryopreservation process. In the first experiment, percentages of motile spermatozoa and fertilizing capacities of spermatozoa were evaluated when mouse spermatozoa were cryopreserved using three previously reported cryopreservation media: (1) 18% raffinose in 3% skim milk; (2) Tes/Tris medium containing 25% egg yolk and 1.25% glycerol; and (3) PBS containing 18% raffinose and 1.75% glycerol, each at three different cooling rates (-3, -10, and -50 degrees C/min). Spermatozoa frozen in the skim milk/raffinose medium exhibited the highest percentage of motile spermatozoa (39%) when cells were frozen at -10 degrees C/min (P<0.05). The second experiment evaluated the effects of modifying the Tes/Tris/egg yolk medium, comparing different concentrations of egg yolk, BSA, and sodium dodecyl sulfate. Reducing egg yolk from 25% of the medium volume to 5%, increased percentages of motile spermatozoa after cryopreservation from 29 to 36% (P<0.05). Addition of 1% BSA and sodium dodecyl sulfate to medium containing 5% egg yolk further improved percentages of motile spermatozoa after freezing. In the final experiment, 20% whole egg was substituted for 5% egg yolk and 1% BSA used in previous experiments and resulted in percentages of motile spermatozoa (51%) equal to that of the skim milk-raffinose medium. However, fertility rates were higher (68%) than for spermatozoa frozen in the skim milk-raffinose medium (P < 0.05) and were comparable to the fertility rates of fresh spermatozoa (77%; P>0.05). In conclusion, freezing mouse spermatozoa in a medium containing 20% whole egg, 0.035% sodium dodecyl sulfate, and 1.25% glycerol using a cooling rate of -10 degrees C/min preserves the motility and fertilization capacity of mouse spermatozoa.     

In vitro embryo production in camel (Camelus dromedarius) from in vitro matured oocytes fertilized with epididymal spermatozoa stored at 4 degrees C

Experiments were conducted to study the effect of storing epididymal spermatozoa, in tris-tes- and tris-lactose egg yolk extenders, on their fertilizing ability and subsequent in vitro embryo development. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 degrees C and on ice (0-1 degrees C), respectively. Cumulus oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25COCs/well) containing 500 microL of maturation medium and cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in air for 36 h. Spermatozoa were collected from the cauda epididymides in syringes containing 2-3 mL of either tris-tes- or tris-lactose egg yolk extender. They were cooled down slowly and stored at refrigeration (4 degrees C) temperature. The spermatozoa were evaluated for motility and used for IVF of IVM oocytes on the day of collection and after 2, 4, 6 and 8 days of storage. On the day of IVF, spermatozoa were prepared by the swim up technique and both spermatozoa and oocytes were co-incubated at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air for 15-16 h. Presumptive zygotes were either fixed and stained with Hoechst 33342 for evaluation of fertilization or were cultured in 500 microL of the culture medium at 38.5 degrees C in an atmosphere of 5% CO(2), 5% O(2) and 90% N(2) in air. There was no significant difference (P>0.05) in the proportion of oocytes fertilized with spermatozoa stored in either of the two extenders for up to 8 days. The proportion of oocytes that cleaved (43-60%) and those that developed to blastocysts (14-21%) did not show any difference (P>0.05) either, when spermatozoa from different days of storage were used. First cleavage was observed as early as 16 h after IVF, early blastocysts had developed by day 4, expanded blastocysts after day 5 and hatching of blastocysts started after day 6 of culture. It may be concluded that dromedary epididymal spermatozoa survive in storage for at least 8 days in tris-lactose- and tris-tes egg yolk diluents at 4 degrees C. These spermatozoa maintain fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures.     

Liposomes as an alternative to egg yolk in stallion freezing extender

Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these liposomes. In Experiment 2, post-thaw motility and membrane integrity of spermatozoa were also analyzed. Experiment 1 demonstrated that liposomes composed of phospholipids E80 (commercial lecithins from egg yolk composed mainly of phosphatidylcholine and phosphatidylethanolamine) and of Hank's salts-glucose-lactose solution (E80-liposomes) were the most efficient in preserving post-thaw motility. The optimal concentration was 4 % (v/v). In Experiment 2, fertility rate after artificial insemination of semen frozen with E80-liposomes was 55 % (22/40) compared with 68 % (27/40) with the control extender containing egg yolk (EY) (p = 0.23). Post-thaw motility parameters were higher with EY than with E80-liposomes (p < 0.0001). For post-thaw membrane integrity no difference was observed between the two extenders (p = 0.08). Liposomes composed of egg yolk phospholipids appeared to be a promising alternative to replace egg yolk in semen freezing extenders in equine species.