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.     

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.     

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.     

The effect of zwitterion buffers on the freezability of Boer goat semen

Twenty semen samples with mass activity greater than +3 were collected from six healthy, mature Boer goat bucks. Each ejaculate was divided into four equal parts and extended at 37 degrees C in Tris, Test, Tes and Bes buffers containing egg yolk and glycerol. Semen was placed into medium size French strawsand after 2 hours of equilibration at 5 degrees C, frozen in the vapour phase and stored in liquid nitrogen for 7 days at -196 degrees C. Progressive motility, the number of live spermatozoa and glutamic oxaloacetic transaminase (GOT) release were studied after the initial extension, after equilibration and after 15 minutes and 7 days of freezing of semen. Semen samples when extended with Tris yolk glycerol showed significantly (P<0.01) higher progressive motility and live spermatozoa than when extended with the other zwitterion buffer-based extenders. The change of extenders did not influence the release of GOT at various stages of freezing of semen (P>0.05).     

Liquid storage of flow cytometrically sorted ram spermatozoa

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.     

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.     

A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles

Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome integrity of bovine sperm processed in egg yolk-based extender by flow cytometer. SYBR-14 and propidium iodide (PI) enabled the discrimination of sperm cells from egg yolk and debris particles, which was instrumental for the flow cytometric analyses of frozen-thawed bovine sperm, because it implied that washing steps to remove egg yolk were no longer required. In addition, phycoerythrin-conjugated peanut agglutinin (PE-PNA) was used to discriminate acrosome-damaged/reacted sperm cells from acrosome-intact cells. Repeatability was calculated using two processed ejaculates of 10 bulls. Three straws per batch were analyzed in duplicate measurements. Method-agreement analysis between the SYBR-14/PE-PNA/PI and fluorescein isothiocyanate (FITC)-conjugated PNA was performed, with FITC-PNA/PI staining being carried out on 14 frozen-thawed semen samples immediately after thawing and after a 3-h incubation at 37 degrees C. The British Standards Institution repeatability index of the SYBR-14/PE-PNA/PI combination was 2.6%. On average, the FITC-PNA/PI method showed a 6.3% overestimation of the live and acrosome-intact sperm cell subpopulation. In conclusion, the new triple-stain combination is highly repeatable and easy to use in routine application, and it provides a more precise estimate for the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.     

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.     

Use of two freezing extenders to cool stallion spermatozoa to 5 degrees C with and without seminal plasma

Motion characteristics of cooled stallion spermatozoa in 2 freezing extenders were studied. Ejaculates from 8 stallions were split into treatments and cooled in thermoelectric cooling units at each of 2 rates. Cooling started at 37 degrees C for Experiments 1 and 3 and at 23 degrees C for Experiments 2 and 4, at a rate of -0.7 degrees C/min to 20 degrees C and from 20 to 5 degrees C, at either -0.05 degrees C/min (Rate I) or -0.5 degrees C/min (Rate II). Percentages of motile (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, 24 and 48 h. Treatments in Experiment 1 were modified skim milk extender (SM); SM + 4% egg yolk (EY); SM + 4% glycerol (GL); and SM + 4% egg yolk + 4% glycerol (EY + GL). At 24 and 48 h, MOT and PMOT were lowest (P < 0.05) for spermatozoa extended in SM + EY; spermatozoa in SM + GL had the highest MOT and PMOT. Thus, glycerol partially protected spermatozoa against the effects of cooling after long-term storage. Treatments in Experiment 2 were SM, semen centrifuged and pellet resuspended in SM (SMc), SM + EY, and semen centrifuged and pellet resuspended in SM + EY (EYc). Spermatozoa in SM + EYc had the highest (P < 0.05) PMOT at 24 h and MOT and PMOT at 48 hours. Spermatozoa in SM + EY (not centrifuged) had the lowest MOT and PMOT at 24 and 48 h, respectively. There was a detrimental interaction between egg yolk and seminal plasma. Extenders in Experiment 3 were Colorado extender (CO3), CO3 + 4% egg yolk (EY), CO3 + 4% glycerol (GL), and CO3 + 4% egg yolk + 4% glycerol (EY + GL). Spermatozoa in CO3 + EY had the lowest (P < 0.05) PMOT at 24 and 48 h. CO3 did not protect spermatozoa cooled in the presence of seminal plasma. Therefore, in Experiment 4 we tested CO3 with seminal plasma present (control) and semen centrifuged and pellet resuspended in CO3 (CO3c), CO3 + EY (EYc), CO3 + GL (GLc) and CO3 + EY + GL (EY + GLc). Spermatozoa in CO3 had the lowest (P < 0.05) MOT and PMOT at all time periods, which suggested a detrimental interaction of this extender with seminal plasma.     

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.     

Factors affecting the removal of low-molecular-weight fractions (LMWF) from egg yolk and seminal plasma in extended semen by dialysis: effect on post-thaw sperm survival

Three factors affecting dialysis of bovine semen were studied. These factors were (1) dialysis rates of egg yolk, seminal plasma, and glycerol, (2) temperature (37 degrees C, 5 degrees C, and while cooling from 37 to 5 degrees C), and (3) dialysis ratios between retentate and dialysate (1:1, 1:10, 1:20, 1:50, and 1:100). Ninety percent of the low-molecular-weight fraction (LMWF) from seminal plasma, egg yolk, and glycerol was removed from the retentate in a 2-hr period at 5 degrees C, and only slight changes were detected after the third hour of dialysis. Temperature affected dialysis and was faster at 37 degrees C. It was also found that a 1:20 dialysis ratio was sufficient to obtain 90% clearance of the LMWF. The effect of sperm dilution ratio, dialysis ratio, and exchange of the LMWF from egg yolk and/or seminal plasma for buffer systems was also studied. An improvement in post-thaw motility of spermatozoa (P less than 0.05) was obtained when the LMWF from both seminal plasma and egg yolk were replaced. A third experiment was conducted to study the effect of different combinations between the buffer systems, TEST and Na citrate, in the dialysate. The results indicated that a 1:1 combination of iso-osmotic solutions (320-325 mOsm/Kg, pH 7.0) between these two buffers, with 5% glycerol (v/v), yielded significant (P less than 0.05) sperm post-thaw motility as compared with the individual use of TEST-glycerol or Na citrate-glycerol. Dialyzed samples also yielded sperm post-thaw motility higher than that of the nondialyzed samples. Colloidal materials in the dialysate did not affect survival of spermatozoa.     

Effect of egg yolk concentration on cryopreserving Spanish ibex (Capra pyrenaica) epididymal spermatozoa

Tris-egg yolk based diluents provide adequate cryoprotection for the sperm of most wild species in which they have been tested. The objective of the current study was to evaluate various Tris-based diluents containing different concentrations of egg yolk, for the fertilizing ability of epididymal spermatozoa of the Spanish ibex (Capra pyrenaica) after freezing and thawing. For this purpose, we used heterologous in vivo fertilization by intrauterine insemination of domestic goats (Capra hircus). In Experiment 1, a Tris-citric acid-glucose (TCG) diluent containing 6% (v/v) egg yolk and a TCG extender containing 20% egg yolk were compared. In Experiment 2, a TCG-6% egg yolk extender was compared with Triladyl-20% egg yolk. Diluted samples were cooled slowly to 5 degrees C over 1 h and equilibrated at that temperature for 2 h. At that point, aliquots of samples were loaded into 0.25 ml straws, and frozen in nitrogen vapor for 10 min. The fertility of spermatozoa frozen in TCG-6% egg yolk was higher (P<0.05) than for those extended with TCG-20% egg yolk, and tended to be higher than for those frozen with Triladyl-20% egg yolk. From the results of this study, the use of Tris-based extenders containing low concentrations of egg yolk (6%) is recommended for cryopreserving Spanish ibex epididymal spermatozoa.     

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.     

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.     

Viability assessment of dog spermatozoa using flow cytometry

The percentages of living and dead spermatozoa in fresh dog semen samples were assessed by means of a dual staining technique using carboxifluorescein diacetate (CFDA) and propidium iodide (PI). Two ejaculates were obtained from dogs, each ejaculate was divided into 4 aliquots, and different proportions of freeze-killed cells were added to each aliquot. Data obtained by flow cytometry analysis of each sample were compared with those obtained by the microscopic evaluation under epifluorescence illumination and by phase-contrast microscopy evaluation of the samples stained with eosin-nigrosin. Regression analysis was used to compare the 3 methods for membrane integrity assessment of canine spermatozoa, and high correlation coefficients were found between the flow cytometry procedure and the 2 microscopy techniques. The results from this study validate the use of flow cytometry as a precise method for assessing the viability of dog spermatozoa.     

Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses

Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained the green fluorescein fluorophore mainly in the acrosome. A third population, presumed to be degenerate spermatozoa, possessed only red fluorescent nuclei. These populations were quantified using dual parameter flow cytometry in 14 samples of cryopreserved bovine spermatozoa for which fertility and seminal quality data were available. Flow cytometric analyses were highly correlated with other seminal quality measurements. Sequential flow cytometric analyses provided the ability to rapidly quantitate changes in specific fluorescently stained populations. The ability to make rapid quantitative measurements should allow development of new and presumably more reliable information on the functional aspects of spermatozoa.     

Influence of the thawing rate on the cryopreservation of semen from collared peccaries (Tayassu tajacu) using Tris-based extenders

The objective was to evaluate the influence of the thawing rate on the quality of frozen-thawed (cryopreserved in Tris-based extenders) semen obtained from collared peccaries (Tayassu tajacu). Semen from 13 sexually mature collared peccaries males were collected by electroejaculation, and evaluated for motility, vigor, sperm viability, membrane integrity, and sperm morphology. Semen was divided in two equal portions: the first was diluted in Tris-fructose and the other in Tris-glucose, with egg yolk (20%) and glycerol (3%) added to each portion. Extended semen was frozen in liquid nitrogen and thawed using two thawing protocols (37 °C for 1 min or 55 °C for 7 s, followed by an additional 30 s at 37 °C). There were no significant differences between the two extenders after extension, chilling, or glycerol addition. After thawing at 37 °C, there were 37.9 ± 4.2% and 28.5 ± 5.1% motile spermatozoa for samples extended in Tris-fructose and Tris-glucose, respectively, with 33.8 ± 3.7% and 28.2 ± 3.5% motile spermatozoa after thawing at 55 °C (no significant differences). Furthermore, there were no significant interactions between extenders and thawing protocols for any semen end point. In conclusion, semen from collared peccaries was successfully cryopreserved in Tris-based extenders and thawed with two protocols (37 °C for 1 min or 55 °C for 7 s).     

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.     

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%.     

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.