Difference In Post-thaw Motility Research Articles

Impact of Cysteine as a Semen Additive on Mitigating the Decline in Sperm Quality due to Summer Stress in Riverine Buffaloes (Bubalus bubalis)

The seasonal variations such as temperature, rain and humid environment greatly influences reproduction, and semen quality particularly in buffalo bulls, either positively or negatively. The heat stress coupled with freezing and thawing processes generates reactive oxygen species (ROS), which further reduce post-thaw spermatozoa quality. Application of cysteine as a semen additive have been reported to alleviate such damaging effect in canine, buck, bull, human, boar, and rooster. Therefore, objective of this study was to investigate ameliorating effect of cysteine on summer stress on buffalo spermatozoa. For the experiment, 24 ejaculates with individual progressive motility >70, volume >1mL and sperm concentration 1000 million/mL from four Murrah buffalo bulls were collected and divided into four groups as Control (Group-I), and Group-II, III, and IV (4, 8, and 12 mmol cysteine, respectively) containing 80 million spermatozoa per mL. Semen was filled (French mini straw), sealed and equilibrated at 40⸰C and frozen into LN2. The group containing L-cysteine at 4 mmol concentration had higher significant (p<0.05) difference in post thaw motility, viability, plasma membrane integrity and acrosome integrity, TAC, DNA integrity and lower significant (p<0.05) difference in spermatozoa abnormality, LPO and intracellular ROS. Moreover, cysteine concentration at 8 mmol has no beneficial effect compared to the Control, whereas Group-IV had greater adverse effect. This may be due to its toxicity at this concentration. It is evident from the study that the cysteine molecules at 4 mmol concentration produced more pronounced improvement in the freezability potential of buffalo bull spermatozoa.

Development of an open hardware 3-D printed conveyor device for continuous cryopreservation of non-batched samples

A great challenge among communities participating in germplasm repository development is to obtain suitable cryopreservation equipment and devices. Commercial programmable freezers are costly and thus unaffordable to many users. Self-made devices have substantial variability , resulting in few opportunities for standardization across communities. The development of open hardware with the increasing accessibility of three-dimensional (3-D) printing offers rapid prototyping and easy fabrication of devices by users around the world at low cost. The present study explored the feasibility of developing operational prototypes of 3-D printed motorized cryopreservation devices for continuous freezing of non-batched samples. A controlled cooling conveyor device (CCCD) was designed and fabricated to cryopreserve sperm samples in straws that were loaded onto chain links suspended over liquid nitrogen held in a Styrofoam box. Cooling rates of 5–34 °C/min for 0.5-mL French straws were produced by adjusting the height of conveyor chains, slopes, and liquid nitrogen mass. The plunge temperature (−47 °C to −61 °C) was controlled by adjustment of conveyor speed. The cooling curves from the CCCD were comparable to a commercial programmable freezer. There were no significant differences in post-thaw motility of sperm from ornamental (Koi) common carp (Cyprinus carpio) among samples frozen with the CCCD and those frozen with a commercial programmable freezer. The post-thaw sperm motility was consistent among samples frozen in the CCCD across a 15-min time span. The CCCD prototypes in the present study proved to be feasible and functional as low-cost, customizable, portable, and yet standardizable options for freezing of individual (non-batched) samples. Additional design alternatives are proposed to facilitate further adaptation and development by diverse user communities.

Corrigendum to: Efficacy of short-term cold storage prior to cryopreservation of spermatozoa in a threatened lizard.

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 25°C or 4°C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 25°C, but did not decrease significantly until 4 days after collection at 4°C, with >30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 4°C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles.

Efficacy of short-term cold storage prior to cryopreservation of spermatozoa in a threatened lizard.

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 25°C or 4°C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 25°C, but did not decrease significantly until 4 days after collection at 4°C, with >30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 4°C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles.

Temporal and Concentration Effects of Methanol on Cryopreservation of Zebrafish (Danio rerio) Sperm.

We evaluated the cryoprotective effects of methanol on zebrafish sperm at different concentrations, exposure times, and stages during cryopreservation. Samples were collected by crushing of dissected testes or abdominal stripping. After exposure to 0%, 2%, 5%, 8%, and 10% methanol for 0-11 min, fresh sperm (1 × 106 cells/mL) did not show changes in plasma membrane integrity (measured by flow cytometer), but cell size changes (light scatter) were observed after exposure to 8% or 10%. After exposure for 0-60 min, fresh sperm (1 × 108 cells/mL) did not show significant changes in survival or membrane integrity. Sperm cryopreserved in 5%, 8%, and 10% methanol showed high post-thaw survival, in 5% and 8% showed high post-thaw motility, and in 5% showed highest post-thaw membrane integrity compared to other concentrations between 0% and 10%. Within 0-60 min after thawing, no significant differences in cell survival and membrane integrity were found for any concentration (p ≥ 0.269). Comparison of 5% and 8% methanol for dissected testes (n = 20) revealed no difference in post-thaw motility, membrane integrity, cell survival, fertilization, or hatching, embryo viability; for stripped sperm (n = 10), no differences were observed in post-thaw membrane integrity, fertilization, and embryo viability, however, higher motility and survival were detected in 5% than in 8% methanol. Thus, a concentration of 5% methanol seems most suitable for cryopreserving zebrafish sperm based on post-thaw survival and motility.

Influence of bull age, ejaculate number, and season of collection on semen production and sperm motility parameters in Holstein Friesian bulls in a commercial artificial insemination centre.

In the current era of genomic selection, there is an increased demand to collect semen from genomically selected sires at a young age. The objective of this study was to assess the effect of bull age, ejaculate number, and season of collection on semen production (ejaculate volume, sperm concentration, and total sperm number; TSN) and sperm motility (prefreeze and post-thaw total and gross motility) parameters in Holstein Friesian bulls in a commercial artificial insemination (AI) center. The study involved the interrogation of a large dataset collected over a 4-yr period, (n = 8,983 ejaculates; n = 176 Holstein Friesian bulls aged between 9 mo and 8 yr). Bulls aged less than 1 yr had the poorest semen production and sperm motility values for all parameters assessed compared with bulls older than 1 yr (P < 0.01). First ejaculates had greater semen production and greater prefreeze motility values than second consecutive ejaculates (P < 0.01), but despite this, there was no difference in post-thaw motility. When subsequent ejaculates were collected from bulls aged less than 1 yr, semen production and sperm motility did not differ compared with mature bulls. Semen collected in winter was poorest in terms of sperm concentration and TSN, but best in terms of post-thaw motility (P < 0.01). In conclusion, second ejaculates can be collected, particularly from bulls aged less than 1 yr, without a significant decrease in post-thaw sperm motility, thus may be a useful strategy to increase semen availability from young genomically selected AI bulls in high demand.

Vitrification as an Alternative Approach for Sperm Cryopreservation in Marine Fishes.

The Southern Flounder Paralichthys lethostigma is a high-value species and a promising aquaculture candidate. Because sperm volume can be limited in this species (<500 μL), new sperm cryopreservation methods need to be evaluated. Vitrification is an alternative to conventional slow-rate freezing, whereby small volumes are cryopreserved at high cooling rates (>1,000°C/min). The goal of this work was to develop a standardized approach for vitrification of Southern Flounder sperm. The specific objectives were to (1) evaluate thawing methods and vitrification solutions, (2) evaluate the postthaw membrane integrity of sperm vitrified in different cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the ability of vitrified sperm to fertilize eggs. From the vitrification solutions tested, the highest postthaw motility (28 ± 9% [mean ± SD]) and membrane integrity (11 ± 4%) was observed for 20% ethylene glycol plus 20% glycerol. There was no significant difference in postthaw motility of sperm thawed at 21°C or at 37°C. Fertilization from vitrified sperm in one trial yielded the same fertilization rate (50 ± 20%) as the fresh sperm control, while the sperm from the other two males yielded 3%. This is the first report of fertilization by vitrified sperm in a marine fish. Vitrification can be simple, fast, inexpensive, performed in the field, and, at least for small fishes, offers an alternative to conventional cryopreservation. Because of the minute volumes needed for ultrarapid cooling, vitrification is not presently suited as a production method for large fishes. Vitrification can be used to reconstitute lines from valuable culture species and biomedical models, conserve mutants for development of novel lines for ornamental aquaculture, and transport frozen sperm from the field to the repository to expand genetic resources.

High-throughput sperm cryopreservation of eastern oyster Crassostrea virginica

Sperm cryopreservation is a valuable tool in germplasm preservation and breeding. Despite many studies, reliable and routine cryopreservation of oyster sperm remains a challenge. The goal of this study was to develop a reliable protocol for sperm cryopreservation of the eastern oyster Crassostrea virginica with high-throughput processing by using two types of 0.5-ml straws (French and CBS™ high security straws). The objectives were to: 1) evaluate the effect of 10% methanol, dimethyl sulfoxide (DMSO), and propylene glycol as cryoprotectants at cooling rates of 5, 20 and 40°C/min from 5 to −80°C and thawing at 30, 40 and 50°C; 2) evaluate the effect of cooling rates of 10, 15, 20, 25 and 30°C/min with 10% DMSO as cryoprotectant and thawing at 40°C; 3) evaluate the effect of equilibration time (10–60min) before freezing; 4) evaluate the effect of sperm concentrations from 1×108 to 1×109 for freezing, and 5) verify the established protocol by freezing sperm from 16 individual males. Among the three cryoprotectants, DMSO yielded the highest post-thaw motility at a cooling rate of 20°C/min when thawed at 30 or 40°C. Further evaluation of cooling rates of 10, 15, 20, 25 and 30°C/min showed that 20 or 25°C/min yielded the highest post-thaw motility (34±5%) and fertility (77±12%) for French straws and CBS straws (28±3% and 69±14%). Equilibration times of 10 to 60min did not cause significant differences in post-thaw motility when freezing with 10% DMSO at a cooling rate of 25°C/min. Also, sperm concentrations ranging from 1×108 to 1×109 at freezing did not cause significant differences in post-thaw motility. Sperm concentration after thawing was not different compared to that before freezing, and no agglutination was observed in the post-thaw samples. Finally, after thawing, sperm cryopreserved from 16 males with this protocol showed 58±24% fertility (from 18 to 86%) for French straws, and 54±21% fertility for CBS straws (from 18 to 95%). Overall, this study provided a reliable protocol for sperm cryopreservation in the eastern oyster with potential for high-throughput processing which can produce thousands of straws per day with homogenous and reliable quality.

High-throughput cryopreservation of spermatozoa of blue catfish ( Ictalurus furcatus): Establishment of an approach for commercial-scale processing

Hybrid catfish created by crossing of female channel catfish ( Ictalurus punctatus) and male blue catfish ( Ictalurus furcatus) are being used increasingly in foodfish aquaculture because of their fast growth and efficient food conversion. However, the availability of blue catfish males is limited, and their peak spawning is at a different time than that of the channel catfish. As such, cryopreservation of sperm of blue catfish could improve production of hybrid catfish, and has been studied in the laboratory and tested for feasibility in a commercial dairy bull cryopreservation facility. However, an approach for commercially relevant production of cryopreserved blue catfish sperm is still needed. The goal of this study was to develop practical approaches for commercial-scale sperm cryopreservation of blue catfish by use of an automated high-throughput system (MAPI, CryoBioSystem Co.). The objectives were to: (1) refine cooling rate and cryoprotectant concentration, and evaluate their interactions; (2) evaluate the effect of sperm concentration on cryopreservation; (3) refine cryoprotectant concentration based on the highest effective sperm concentration; (4) compare the effect of thawing samples at 20 or 40 °C; (5) evaluate the fertility of thawed sperm at a research scale by fertilizing with channel catfish eggs; (6) test the post-thaw motility and fertility of sperm from individual males in a commercial setting, and (7) test for correlation of cryopreservation results with biological indices used for male evaluation. The optimal cooling rate was 5 °C/min (Micro Digitcool, IMV) for high-throughput cryopreservation using CBS high-biosecurity 0.5-ml straws with 10% methanol, and a concentration of 1 × 10 9 sperm/ml. There was no difference in post-thaw motility when samples were thawed at 20 °C for 40 s or 40 °C for 20 s. After fertilization, the percentage of neurulation (Stage V embryos) was 80 ± 21%, and percentage of embryonic mobility (Stage VI embryo) was 51 ± 22%. There was a significant difference among the neurulation values produced by thawed blue catfish sperm, fresh blue catfish sperm ( P = 0.010) and channel catfish sperm ( P = 0.023), but not for Stage VI embryos ( P ⩾ 0.585). Cryopreserved sperm from ten males did not show significant variation in post-thaw motility or fertility at the neurulation stage. This study demonstrates that the protocol established for high-throughput cryopreservation of blue catfish sperm can provide commercially relevant quantities and quality of sperm with stable fertility for hybrid catfish production and provides a model for establishment of commercial-scale approaches for other aquatic species.

Differences in post-thaw motility of spermatozoa between land-locked and amphidromous forms of ayu ( Plecoglossus altivelis)

There are two types of ayu fish, a land-locked and an amphidromous form, and the former usually matures about 1–2 months earlier than the latter. We compared sperm motility before and after cryopreservation during the spawning season for 2 years to determine whether there are any differences in the tolerance of sperm cells to cryopreservation between the two forms. Milt was diluted with 10% methanol and 90% fetal bovine serum, dispensed into acrylic capillary tubes, cooled in liquid N 2 (LN) vapor to − 50 °C at a cooling rate of − 42.5 °C/min, and plunged into LN for storage. In both 2002 and 2003, the post-thaw motility of the spermatozoa obtained from mature land-locked form males was low throughout their spermiation period, while that of amphidromous form males increased considerably. The mean percent motility of the spermiation period was significantly different between the two types of ayu in both years. Furthermore, we compared the post-thaw motility of spermatozoa of the two forms at the peak of their spermiation periods in 2007. Although the percent motility of fresh milt was high in both forms, the post-thaw motility of spermatozoa in the land-locked form was significantly lower than that of the amphidromous form. These results indicate that the tolerance of ayu spermatozoa to cryopreservation differs between the land-locked and amphidromous forms.

Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters, Crassostrea gigas

The availability of tetraploid Pacific oysters provides a unique opportunity for comparative studies of sperm cryopreservation between diploids and tetraploids. In parallel to studies with sperm from diploid oysters, this study reports systematic factor optimization for sperm cryopreservation of tetraploid oysters. Specifically, this study evaluated the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), and straw size. Similar to sperm from diploids, the optimal cooling rate was 5 °C/min to −30 °C, followed by cooling at 45 °C/min to −80 °C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations showed that a combination of the cryoprotectants 6% polyethylene glycol/4% propylene glycol and 6% polyethylene glycol/4% dimethyl sulfoxide yielded consistently high post-thaw motility. A long equilibration (60 min) yielded higher percent fertilization, and confirmed that extended equilibration could be beneficial when low concentrations of cryoprotectant are used. There was no significant difference in post-thaw motility between straw sizes of 0.25 and 0.5 mL. Despite low post-thaw fertilization (<10%) in general for sperm from tetraploids, optimized protocols in the present study effectively retained post-thaw motility for sperm from tetraploid oysters. This study confirmed that sperm from tetraploid Pacific oysters were more negatively affected by cryopreservation than were those of diploids. One possible explanation is that sperm from these two ploidies are different in their plasma membrane properties (e.g., structure, permeability, and elasticity), and the plasma membrane of sperm from tetraploids is more sensitive to cryopreservation effects. The fact that combinations of non-permeating and permeating cryoprotectants improved post-thaw motility in sperm from tetraploids provided presumptive evidence for this interpretation.

Studies on the Effect of Supplementing Boar Semen Cryopreservation Media with Different Avian Egg Yolk Types on in Vitro Post‐thaw Sperm Quality

Fertility after insemination of cryopreserved boar semen is currently below that of fresh semen. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using an adapted Westendorf method in which the chicken egg yolk was replaced by either duck or quail egg yolk. The different composition of the yolk types, particularly the amount of cholesterol, fatty acids and phospholipids, were thought to potentially afford a greater level of protection to sperm against damage during freezing and thawing. Sperm frozen in medium containing chicken egg yolk displayed higher motility immediately after thawing, but there was no difference in the motility of sperm frozen with different types of egg yolk 3 or 6 h after thawing and maintenance at 37 degrees C. Sperm frozen in media containing chicken or duck egg yolk had a higher proportion of intact acrosomes immediately after thawing than sperm frozen in medium containing quail egg yolk, but 6 h after thawing and maintenance at 37 degrees C the sperm that had been frozen in medium containing chicken egg yolk had a higher proportion of intact acrosomes than the sperm frozen in media containing duck or quail egg yolk. Analysis of the composition of the different yolk types showed that the basic components of the yolks were similar, but the ratios of fatty acids and phospholipid classes differed. Duck egg yolk had more monounsaturated fatty acids (MUFA) than chicken egg yolk, which had more MUFA than quail egg yolk. Duck egg yolk contained more phosphotidylinositol (PI) than chicken or quail egg yolks and quail egg yolk contained more phosphotidylserine than either chicken or duck egg yolks. The differences in post-thaw motility and acrosome integrity of boar sperm when frozen in media containing the different types of egg yolk may be due to the variation in composition.

Effect of sugar type on the survival of frozen-thawed rhesus monkey (Macaca mulatta) sperm

Sperm-freezing extenders supplemented with sugar or a combination of different sugars are widely used for the cryopreservation of nonhuman primate spermatozoa. Understanding which sugar or combination of sugars offers the highest level of cryoprotection would be beneficial for the development of sperm-freezing extenders. In the present study we aimed to investigate the effect of glucose, lactose, and raffinose separately or in combination on the cryosurvival of rhesus monkey spermatozoa. Toward that end, we prepared eight extenders by adding various types of sugars to a basic medium (BM): G-BM (0.3 M glucose), L-BM (0.3 M lactose), R-BM (0.3 M raffinose), LG-BM (0.15 M lactose+0.15 M glucose), RG-BM (0.15 M raffinose+0.15 M glucose), LR-BM (0.15 M lactose+0.15 M raffinose), and LRG-BM (0.1 M lactose+0.1 M raffinsoe+0.1 M glucose). A saline control (0.157 M sodium chloride) was also used. The results showed no significant difference in post-thaw motility when spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRG-BM, but the post-thaw motility was significantly lower when it was frozen with R-BM, LR-BM, and the saline control. The highest plasma membrane integrity was achieved when spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRG-BM, and the highest acrosome integrity was achieved with G-BM, L-BM, LG-BM, RG-BM, LRG-BM, and the saline control. The results indicate that the various sugars offered different protective effects. For the cryopreservation of rhesus monkey spermatozoa, glucose (monosaccharide) and lactose (disaccharide) were shown to be more suitable than raffinose (trisaccharide) for preserving spermatozoal motility, plasma membrane, and acrosome. Specifically, raffinose was detrimental to sperm acrosome integrity.

Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 °C/min to −30 °C followed by cooling at 45 °C/min to −80 °C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.

The development of a sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum): evaluation of cryoprotectants and diluents

Three cryoprotectants (dimethyl sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based and saline based) were mixed (9 parts diluent–1 part cryoprotectant) factorially to produce six extenders that were tested to develop an effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum). Sperm were diluted 1:3 with each extender and frozen by flotation on liquid nitrogen before being submerged and stored for 30 days. Sperm left unfrozen in each extender for 20 min showed no toxic effects on motility. Extenders containing propylene glycol (PG) as cryoprotectant yielded higher post-thaw sperm motilities than those containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose-based diluent performed better than the saline-based diluent when DMSO was used as cryoprotectant, but there were no differences in post-thaw motility between diluents for the other cryoprotectants. Activating sperm with ovarian fluid and sea water instead of sea water alone had no effect on post-thaw motility. In fertilization trials, no differences were observed between any of the extenders and fresh milt when milt, eggs and sea water were left in contact for 1 h. When sperm were forced to compete for eggs by reducing contact time to 20 s, fertilization results followed those of sperm motility rates. Percentage hatch and morphology of larvae at hatching did not differ for eggs fertilized by cryopreserved and fresh sperm. This study represents the first reported successful attempt at cryopreserving winter flounder sperm and should improve gamete and broodstock management protocols for this species.

Comparison of two processing systems for bull semen with regard to post-thaw motility and nonreturn rates

Two semen processing systems based on either skim milk-yolk extender or Biladyl® were compared with regard to post-thaw motility and nonreturn rates. The skim milk-yolk extender contained penicillin and streptomycin, while Biladyl® consisted of tylosin, gentamycin, spectinomycin and lincomycin. The dilution of semen involved a 2-step procedure for both extenders. The second dilution with the skim milk-yolk extender took place at 5 °C, while the Biladyl® extender was processed at room temperature. In this study 197 ejaculates from 45 Norwegian Cattle bulls were used. The ejaculates were split-sampled and diluted with the 2 extenders. A total of 41,445 first inseminations was recorded, 21,035 with semen diluted with skim milk-yolk and 20,410 with Biladyl®. The 60-d nonreturn rates were 73.1 and 71.9% for skim milk-yolk and Biladyl®, respectively. The results for skim milk-yolk were significantly better than for Biladyl® (P < 0.01). No difference in post-thaw motility for these extenders was found.

The cryopreservation of donor semen by a simplified method: use in an IVF and GIFT programme

The cryopreservation of semen used in assisted reproduction procedures was carried out exclusively by a simplified method in which a mixture of semen and cryoprotectant was contained in 1-ml tuberculin syringes and plunged directly into liquid nitrogen. Donor semen samples halved and frozen in syringes and in straws in a controlled-rate freezer showed no significant difference in post-thaw motility (P = 0.217) or survival (P = 0.217) after 30 min. However, after 180 min the survival rate showed a significant reduction in syringes (P = 0.045). A significant difference (P less than 0.00008) in the rate of fertilization of oocytes was seen in IVF cycles using frozen-thawed donor sperm (58/142, 42%) when compared to fresh sperm from husbands (2315/3926, 59%). A significant reduction (P less than 0.00005) in fertilization rate was also observed in the case of supernumerary oocytes in GIFT cycles with the cryopreserved donor sperm (29/132, 22%) compared to the husbands' sperm (239/514, 46%). However, the pregnancy rate following IVF and embryo replacement was the same after fertilization with fresh sperm (75/351, 21%) as opposed to frozen sperm (3/14, 21%). Furthermore, a higher pregnancy rate was observed in GIFT with frozen donor sperm (9/19, 47%) than with fresh sperm from husbands (28/103, 27%), though this was not statistically significant (P = 0.079). These results show this simplified methods of semen cryopreservation to be effective when used in an IVF and GIFT programme, giving pregnancy rates comparable to fresh normospermic semen samples. The method is simple, quick and inexpensive.