Chilled storage of semen from Atlantic halibut, Hippoglossus hippoglossus L. II: effect of spermiation advancement, catheterization of semen, and production-scale application

A method for in vitro storage of Atlantic halibut Hippoglossus hippoglossus L. semen can facilitate seed production. The study aimed at determining the effect of male spermiation advancement on viability of chilled stored spermatozoa. The use of catheterization of semen from the sperm duct was examined. Also, large volumes of semen were stored under sub-optimal production-like conditions in order to determine the suitability of the method into practical use. Semen was collected from two broodstocks: natural photoperiod males, being at the first phase of the reproductive season and 3-month advanced photoperiod males, being at the end of the reproductive season. Semen samples were diluted 1:5 (v/v) with modified Hanks' Balanced Salt Solution supplemented with antibiotics, and stored in Ziploc bags filled with air. Sperm motility parameters, assessed by computer-assisted sperm analysis (CASA), were assessed weekly. Experimental and production-scale fertilization trials were performed. Sperm samples from natural photoperiod males showed significantly longer viability under in vitro storage conditions than sperm from advanced photoperiod males. In the natural photoperiod group, the decrease in spermatozoa motility, curvilinear velocity and straight-line velocity occurred on day 50, 14, and 28 of storage, respectively. Spermatozoa from one of five males were still motile on day 80 of storage, and fertilization rates and embryo survival rates obtained using semen stored for 70 days did not differ from control values and they were significantly higher than values obtained with the use of fresh semen of the same male, but being at the end of reproductive season. Catheterization of semen showed no advantage to stripping the semen without a catheter, even for samples stored undiluted for 1 day of collection, before dilution. Under sub-optimal conditions, spermatozoa stored in large volumes (10-100mL of diluted semen) without any special treatments except for weekly swirling, remained viable for more than 1 month. Production-scale fertilizations with samples stored for 5-21 days resulted in high survivals of embryos and hatchlings. Because of its simplicity and efficiency, the method shows a high potential for use in commercial Atlantic halibut farming. It has already been applied to a halibut breeding programme for the next reproductive season at our research station.     

Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa

Protocols for the successful manipulation and preservation of semen in a given species depend upon a fundamental knowledge of how spermatozoa respond to the physicochemical conditions of the extension media; methods developed for the preservation of eutherian spermatozoa may not necessarily be suitable for marsupial semen. The aim of this study was to investigate the effects on koala sperm motility of serial dilution, changes in temperature, diluent pH and osmolality to establish the optimal physicochemical conditions for short-term semen storage. This study showed that electroejaculated koala semen diluted 1∶1 (v/v) with PBS frequently coagulated after incubation at 35 degrees C, but that further dilution and incubation resulted in a corresponding increase in the percentage of spermatozoa swimming in a non-linear trajectory. The effect of rapid temperature change on the motility of koala spermatozoa was investigated by exposing semen, initially diluted at 35 degrees C, to temperatures of 45, 25, 15 and 5 degrees C. Although sperm motility was reduced after incubation at 45 degrees C, a rapid decrease in temperature of up to 20 degrees C did not result in a significant reduction in sperm motility. However, contrary to evidence in other marsupials, there was a small but significant decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 degrees C. The effects of diluent pH and osmolality on the motility of koala spermatozoa were investigated. These experiments indicated that diluents for koala sperm manipulation should buffer in a pH range of 7-8 and have an osmolality of approximately 300 mmol kg(-1). The final experiment compared the relative effectiveness of Tris-citrate buffer (1% glucose) and PBS to maintain koala sperm motility over a range of incubation temperatures (5-35 degrees C) for up to 8 days. Reduction in sperm motility was directly related to temperature, and motility was sustained for the longest duration when stored at 5 degrees C. The Tris-citrate buffer solution was superior to PBS as a preservation diluent at all temperatures, and koala spermatozoa remained motile even after 42 days storage at 5 degrees C. Spermatozoa diluted in PBS (with Ca(2+) or Mg(2+)) and cooled to 5 degrees C showed evidence of an unusual motility pattern, similar to that of hyperactivated eutherian spermatozoa. This study showed that koala spermatozoa respond to different physicochemical conditions associated with short-term liquid storage in essentially the same way as the spermatozoa of eutherian mammals, although koala spermatozoa appear to be more tolerant of rapid temperature shock. The results of this study can be used to make informed selections with regard to appropriate diluent composition and improved short-term sperm preservation protocols and represent the first such database for any species of marsupial.     

Quantitative characteristics of Atlantic halibut, Hippoglossus hippoglossus L., semen throughout the reproductive season

The overall objective of the study was to investigate changes in quantitative parameters of Atlantic halibut (Hippoglossus hippoglossus L.) semen throughout the reproductive season in order to systematize the knowledge about biology of Atlantic halibut spermatozoa. Semen samples were collected from February to May from broodstock males kept under either a natural or 3-month advanced photoperiod regime. Spermatozoa concentration, semen pH and osmolality, as well as spermatozoa motility parameters were investigated. The use of catheterization of sperm was examined. Also, fertilization tests were performed. We found that spermatozoa concentration increases in a linear-like mode towards the end of the spawning season, which correlated with a decrease in a number of spermatozoa motility parameters, including actual percentage of motile spermatozoa (MOT), curvilinear velocity (VCL) and straight-line velocity (VSL) of spermatozoa. A breakpoint in MOT occurred when spermatozoa reached a concentration in the range of 17-20 x 10(9) spermatozoa/mL. The fertilization ability of sperm from males kept under natural photoperiod decreased in April. Survival of embryos at 80 degrees days produced by fertilizing eggs of single female with sperm from natural photoperiod males was 88, 76 and 41% on April 09 and 17, and May 01, respectively, whereas using sperm from 3-month delayed photoperiod males for fertilizing eggs from the same female on April 27 resulted in 80% of surviving embryos, not differing significantly from the data from April 09. Physical decomposition of spermatozoa was observed towards the end of the season and it was related to an increase in the whole semen osmolality. Catheterization of semen did not improve spermatozoa motility parameters, however, it reduced the variation in recorded values, especially in the case of pH, caused by contamination with feces or urine. Post-seasonal decrease in spermatozoa concentration was likely related to intensive ageing processes. Based on the present study and available data by other researchers, a model of changes of quantitative parameters in Atlantic halibut semen throughout the reproductive season is proposed.     

Artificial insemination of sheep: Effects on fertility of number of spermatozoa inseminated and of storage of diluted semen for up to 18 hours at 5°C

Ram semen was prepared in a buffered glucose-saline solution containing 3% (v/v) egg yolk so that insemination doses of 25 or 100 million spermatozoa in volumes of 50 or 250 μl could be given per ewe at artificial insemination (AI). Fertility was significantly reduced by dilution and, within the treatments of diluted semen, significantly higher lambing rates followed the use of doses of 100 million spermatozoa. The volume of the AI dose had no significant effect on fertility.Of 945 inseminations performed using diluted semen, 388 were with samples that had been cooled to 5°C and stored chilled for 5 or 18 hr. The mean lambing result of 40% for freshly diluted semen was significantly higher than 31.6% and 30.2% for samples stored chilled for 5 and 18 hr respectively. Ewes inseminated with doses of chilled semen containing 25 million spermatozoa had a low lambing rate of 21.3%. The presence of 7.5% glycerol (v/v) in the diluent did not significantly affect the fertility of chilled semen.     

Semen characteristics of the captive Indian white-backed vulture (Gyps bengalensis)

The present paper describes, to our knowledge for the first time, the successful collection and evaluation of semen from the Indian white-backed vulture (Gyps bengalensis), a critically endangered bird. Over a period of 2 yr, semen was collected using the manual massage method and evaluated for semen volume, semen pH, sperm concentration, percentage normal/abnormal spermatozoa, and percentage motile spermatozoa. It appears that the concentration of spermatozoa and percentage motile spermatozoa in the Indian white-backed vultures are low compared to those in other birds. Tyrode medium supplemented with albumin, lactate, and pyruvate (TALP) proved to be the best semen extender compared to two others (Beltsville Poultry Semen Extender and Lake diluent). Furthermore, TALP with 20% egg yolk and supplemented with 8% dimethyl sulfoxide maintained 50% of the initial percentage of motile spermatozoa following cryopreservation and thawing. A computer-aided semen analysis indicated that the spermatozoa of the Indian white-backed vulture are extremely active and swim in linear trajectories for up to 5 h following dilution in TALP. The trajectories were linear with time, but we noticed a decrease in the velocity parameters (average path velocity, curvilinear velocity, and progressive velocity). Thus, the present study provides baseline data on semen characteristics of the highly endangered Indian white-backed vulture, and these data could be of immense importance to reproductive and conservation biologists attempting to breed these animals in captivity, which to date has not been achieved.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

The effect of viscosity of semen diluents on motility of bull spermatozoa

The effect of egg yolk extender on semen viscosity and bull sperm motility of fresh and cooled or deep frozen semen was determined by a computer-assisted system. Viscosity of the extender was determined by flow time. Based on the sperm velocity (velocity of the average path), individual spermatozoon were classified into groups of progressively motile (>==30 microm/sec) and immotile (<10 microm/sec) spermatozoa. The average velocity of progressively motile spermatozoa (VPM), the velocity of linear progressively motile spermatozoa (VLP) and the percentage of linear swimming spermatozoa (LIN) were evaluated. The addition of 10, 20 or 30% egg yolk to Tris buffer (pH 6.5) resulted in a linear decrease of VPM and a decrease in the percentage of progressively motile spermatozoa, but it increased the relative rate of LIN in fresh diluted semen. Increasing the levels of egg yolk in the diluent resulted in higher viscosity. The VLP was significantly higher than the VPM. In refrigerated or frozen semen samples, extender with 30 and 20% egg yolk had a similar effect on the VPM but not on the percentage of progressively motile sperm cells. Freezing of egg yolk (30%) extender to -20 degrees C resulted in a significant increased flow time and higher viscosity. Dilution of semen samples with high viscosity extender decreased the VPM in fresh and chilled semen. Freezing semen of high viscosity extender with glycerol had no apparent effect on the percentage of progressively motile spermatozoa compared with that of non-glycerinated egg yolk extender. The results suggest that different concentrations of egg yolk in the extender can influence the parameters of semen viscosity and sperm motility evaluated by a computer-assisted system.     

Effect of bovine serum albumin on motility and fecundity of turkey spermatozoa before and after storage.

Motility characteristics of turkey spermatozoa before and after storage for 24 h at 7 degrees C in diluent with and without bovine serum albumin (BSA; 1% final concentration) were measured by computer-assisted semen analysis. BSA significantly increased the percentage of motile spermatozoa and sperm velocity, linearity, lateral head displacement and beat frequency in each treatment, but BSA in fresh or stored semen in diluent did not augment hen fertility over 15 weeks of egg production. Fatty-acid-free BSA, globulin-free BSA and Fraction V BSA all significantly increased each sperm motility characteristic compared with semen in diluent alone. The lack of correlation between sperm motility and fecundity emphasizes the need to develop procedures for semen evaluation that accurately predict the fertilizing capacity of an aliquot of semen.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.     

A non-activating diluent to prolong in vitro viability of Apis mellifera spermatozoa: Effects on cryopreservation and on egg fertilization

A non-activating semen diluent does not cause motility or acrosomal reaction or capacitate the sperm cell. The effects of such a diluent on the viability of honey bee spermatozoa stored in ambient conditions were assessed 60 days pre-cryopreservation and 24 h post-cryopreservation. Seven variations of a Tris-based non-activating diluents (FEM1 – FEM7) were compared to samples treated with conventional activating diluent and untreated semen. Semen viability (membrane integrity) was assessed after short- and long-term storage at 14.0 ± 0.2 °C. The non-activating medium FEM7 contained more viable spermatozoa than the activating medium, 24 h after cryopreservation (67.6 ± 10.9% and ~4%, respectively). After 60 days, 22.0 ± 7.8% of spermatozoa was viable in non-activating medium versus 0.0 and 60.8 ± 12.3%, in conventional media and untreated controls, respectively. Hence FEM7 was used to cryopreserve bee semen and subsequently inseminate honey bee queens. The quality of brood produced by the queens was assessed 30–60 days after insemination. The percentage of worker-bee offspring (produced from successfully fertilized eggs) was ~75% for both the non-activating medium and the conventional extender medium. Our results indicate that a non-activating medium possesses significant advantage over the conventional activating medium if the semen requires storage after treatments such as cryopreservation. The percentage of female offspring (from fertilized eggs) produced by queens inseminated with semen diluted in either the activating or non-activating medium did not differ from one another.     

Semen cryopreservation in Bactrian camel (Camelus bactrianus) using SHOTOR diluent: effects of cooling rates and glycerol concentrations

Experiments were conducted with a final goal of providing a suitable protocol for cryopreservation of Bactrian camel semen. In Experiment I, the effect of average cooling rate (slow cooling: 0.14 versus fast cooling: 0.55 degrees C/min) on the viability of chilled semen was evaluated. In Experiment II, the effect of different concentrations of glycerol (4, 6 and 8%) on the post-thaw viability of frozen sperm was investigated. In Experiment III, the efficiency of SHOTOR diluent was compared with IMV buffers for the cryopreservation of camel semen. Viability parameters including progressive forward motility (PFM), plasma membrane integrity and percentage of live spermatozoa were assessed. Progressive forward motility of sperm cooled at the faster rate was superior after incubating for 24h at 4 degrees C compared to that cooled at the slower rate (P<0.05). Post-thaw viability of Bactrian camel sperm was better using a final glycerol concentration of 6% compared to 4 and 8% (P<0.05). Progressive forward motility of frozen-thawed sperm was greater using SHOTOR diluent (29.9%) compared to IMV buffers (4.2%, P<0.05). In conclusion, semen cryopreservation in Bactrian camel is feasible when it is extended in SHOTOR diluent, cooled within 1h (average cooling rate: 0.55 degrees C/min) to 4 degrees C, and then exposed to glycerol, at the final concentration of 6%.     

Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes

The post-thaw survival and fertility of ram spermatozoa frozen in pellets, 0.25- and 0.5-ml PVC straws, and 0.25-ml minitubes were examined. In 5 experiments, a freezing height of 6 cm above the level of liquid nitrogen was optimal for 0.25- and 0.5-ml straws, whereas 4 cm was best for the 0.25-ml minitubes. Post-thaw motility of spermatozoa was lower for semen frozen in straws and minitubes than in pellets (Experiment 1: 43.7 vs 53.4%, P < 0.001), but after freezing was better in 0.5-ml straws and 0.25-ml minitubes than in 0.25-ml straws (Experiment 1: 44.9 vs 41.3%, P < 0.05; Experiment 2: 49.6 vs 46.8%, P < 0.01). Sperm motility was also better for 1:8 (semen:diluent) pre-freezing dilution rate (50.5%) than for 1:4 (45.6%, P < 0.01) and 1:2 (39.8%, P < 0.001) but not the 1:16 (49.5%) dilution rate. Dry ice was a better freezing medium than liquid nitrogen vapor (49.2 vs 46.9% motile spermatozoa, P < 0.001). The post-thaw motility of spermatozoa was similar for the three freezing packages if the semen was loaded at 5 degrees C, but motility was poorer for semen loaded into 0.25-ml straws than 0.25-ml minitubes at 30 degrees C (P < 0.05). In a fertility test, pregnancy rates were influenced by rams (3 rams, P < 0.05) and freezing package (pellets vs 0.25-ml minitube vs 0.25-ml straw vs 0.5-ml straw, P < 0.05) but not freezing medium (liquid nitrogen vapor vs dry ice). More ewes were pregnant after insemination with pellet-frozen semen (106/150, 71%) than with semen frozen in 0.25-ml straws (85/150, 57%; P < 0.05) and in 0.5-ml straws (83/150, 55%; P < 0.01) but not minitubes (98/150, 65%). It was concluded that minitubes provide a useful alternative to pellets as a storage package for ram spermatozoa, allowing for individual dose identification and easier storage while maintaining a fertility rate indistinguishable from that obtained with pellet-frozen semen.     

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.     

Morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees C

Swelling of the apical ridge and anterior acrosome of motile bovine spermatozoa was observed during in-vitro storage using differential interference-contrast optics. This morphological alteration is different from that described as the false acrosome reaction on immotile spermatozoa, apparent in ageing semen samples and which has been associated with cell death. In this study, transmission electron microscopy revealed that the apical ridge acrosomal matrix was extended into complex folds and/or projections. Acrosomal and plasma membrane integrity was retained. Storing spermatozoa (1500 X 10(6)/ml) in seminal plasma at 4 degrees C for 1 day was most conducive to the swelling of the apical ridge. Replacing seminal plasma with egg yolk-citrate inhibited swelling. However, incubating semen at 37 degrees C in egg yolk-Tris-fructose extender (25 X 10(6) spermatozoa/ml) after storage in egg yolk-citrate at 4 degrees C for greater than or equal to 3 days restored the swelling characteristic.     

Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen.

The influence oftemperature, addition of glycerol, initial freezing temperature, method of dilution, level of glycerol in the diluted semen, equilibration time and type of diluent on the survival and fertilizing capacity of deep-frozen according to the best conditions was compared with that of "fresh" semen. The addition of glycerol at plus30 degrees C resulted in a highly significant decrease in the mean proportion of motile spermatozoa immediately after thawing compared with the effect of addition at plus 4 degrees C. The immersion of the straws at minus55 degrees C significantly reduced the revival of the spermatozoa compared with initial freezing at lower temperatures. The exposure time to glycerol had no significant effect on the survival of spermatozoa after thawing and incubation, but fertility was significantly higher with 4% than with 2% glycerol. The I. N. R. A. diluent provided better sperm survival and a significantly higher conception rate than did lactose-egg yolk extender. The semen frozen according to the best conditions (about 50% of the samples) had a fertilizing ability similar to that of "fresh" semen when the proportion of motile spermatozoa before, and after 1 or 3 hr of incubation was equal to or above 45, 40 and 30% respectively.     

The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa

Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even after a rapid decrease in glycerol concentration such as that which accompanies insemination or dilution of semen for assessment of motility.     

Long-term motility and fertility conservation of chilled canine semen using egg yolk added Tris-glucose extender: in vitro and in vivo studies

The effects of medium exchange on motility parameters of chilled canine semen preserved in egg yolk Tris-glucose (EYTG) extender were analyzed over a 27-d period. Semen extender was exchanged at three time points (Days 11, 21 and 27) after collection, when motility parameters were demonstrated to significantly decrease from parameters observed at semen preparation (Day 0) or at day of previous extender exchange. In the absence of medium exchanges, motile spermatozoa were observed up to Day 16 (mean +/- S.D. 1.5 +/- 0.3% of motile spermatozoa). A stimulation of the different semen motility parameters was observed after extender exchange. Semen extender exchange at Day 11 allowed conservation of motility until Day 21, compared to 16 d in the absence of extender exchange. At Day 21, when spermatozoa appeared immobile or dead, a second extender exchange was performed, allowing the extension of motility conservation up to Day 27. The third extender exchange, performed at Day 27, was no longer associated with motility stimulation. Glucose content in the medium decreased slowly over time; a concomitant decrease in pH was also observed. No changes in osmolarity were observed over time. To verify the fertility of long-term conserved chilled semen, two groups of 10 bitches were inseminated either once (Group 1) or twice at 48-h intervals (Group 2) intra-vaginally with semen conserved chilled for a mean of 9 +/- 1.8 d. Out of the 10 bitches inseminated once, 5 became pregnant, versus 7 in the group of animals inseminated twice. The present study reports the possibility to extend the conservation of chilled canine semen up to 3 wk with conservation of good fertility for at least 10 d. The role of energetic substrate and pH alteration is postulated and the classically accepted relation of semen motility/viability is raised.     

Comparison of three diluents for the storage of fresh bovine semen

In New Zealand, 95% of the semen used for artificial insemination in cattle is processed as liquid semen. Storage of liquid semen for up to 3 days in Caprogen) diluent enables a 10-fold reduction of the insemination dose, compared to frozen-thawed semen, without a reduction in fertility. In this Caprogen) diluent spermatozoa are stored under N2 gas in the presence of catalase. However, a new diluent (CEP-2), which was originally based on the biochemical composition of bovine cauda epididymal plasma, could become an appropriate alternative to Caprogen. In this study, the effect of addition of catalase to bovine spermatozoa stored for 6 days in CEP-2 diluent under aerobic and anaerobic conditions was evaluated and compared with a Tris diluent. Additionally, the quality and in vitro fertilizing capacity of fresh bovine semen stored for 6 days at 5 degrees C in the Triladyl, CEP-2 (without catalase and N2 gas) and Caprogen diluent were compared. Addition of 4.5 mg/mL catalase to CEP-2 diluent under aerobic and anaerobic conditions had no effect on sperm quality. Spermatozoa stored in CEP-2 diluent moved faster and straighter than spermatozoa stored in Triladyl or Caprogen diluent. The in vitro fertilization and polyspermy rates did not differ significantly between spermatozoa stored for 6 days at 5 degrees C in CEP-2 and Caprogen diluent, but were significantly lower for spermatozoa stored in Triladyl diluent. We can conclude that based on the in vitro results, the CEP-2 diluent is a better diluent than Triladyl and a good alternative to the Caprogen diluent for long term storage of fresh bovine semen at 5 degrees C. To confirm these promising in vitro results further in vivo experiments are required.     

Measurement of the sperm motility index via the sperm quality analyzer and its relationship to other qualitative sperm parameters

Sperm parameters such as the concentration and percentage of motile spermatozoa are commonly used to assess semen quality. The sperm quality analyzer (SQA) is a device that detects variations in the optical density of motile spermatozoa, providing a sperm motility index (SMI) that is based on various sperm parameters including the concentration, morphology and acrosomal status of motile spermatozoa. The relationship between SMI values of frozen-thawed bovine spermatozoa undergoing swelling in a hypoosmotic medium (100 mOsm/L) and other sperm parameters were evaluated. Frozen semen specimens from 3 bulls were thawed and washed with Ham's F-10 supplemented with 3% BSA and split into 3 (0.2 mL) aliquots. The aliquots were diluted with 1.0 mL of Ham's F-10 (Aliquot 1), isotonic sodium citrate (Aliquot 2), and hypotonic sodium citrate (Aliquot 3). The osmotic pressure of the media used for dilution of Aliquots 1 and 2 was 300 mOsm/L, while that for Aliquot 3 was 100 mOsm/L. Following dilution, the aliquots were incubated for 30 min and manually assessed at 5-min intervals for the percentage and grade of motility (Grades 0 to 4) as well as for the percentage of swollen spermatozoa. Sperm samples were simultaneously evaluated by SQA to obtain the SMI values at the same 5-min intervals during the 30-min incubation. Significant correlations were observed between SMI values and other sperm parameters in Aliquot 3 (P < 0.05). The results indicated that the SMI values obtained from frozen-thawed bovine spermatozoa exposed to a 100 mOsm/L diluent, which causes optimal swelling of spermatozoa, are highly correlated to other sperm parameters. The SQA unit, as applied in this study, can be used for rapid and reliable screening of sperm samples.     

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.