TEST-yolk Buffer Research Articles

Hyaluronan binding assay (HBA) versus sperm penetration assay (SPA). can HBA replace the use of SPA test in male partner screening before in vitro fertilization?

Hyaluronan binding assay (HBA) versus sperm penetration assay (SPA). can HBA replace the use of SPA test in male partner screening before in vitro fertilization?

Positive expression of the immunoglobulin superfamily protein IZUMO on human sperm of severely infertile male patients

Recently, the immunoglobulin superfamily protein IZUMO on the sperm surface was identified as being essential for sperm-egg fusion. Although we examined the expression of IZUMO on sperm of 13 male infertile patients with severe oligozoospermia and/or athenozoospermia and 12 infertile patients with fertilization failure in previous conventional IVF to determine whether it is varied in infertility patients, we could not identify any patients with IZUMO negative by immunocytochemistry.

Slow cooling prevents cold-induced damage to sperm motility and acrosomal integrity in the black-footed ferret (Mustela nigripes)

The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25 degrees C v. 37 degrees C); (3) type of medium (Ham's F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P < 0.05) at 25 degrees C than at 37 degrees C in Ham's or TYB, but temperature did not affect (P > 0.05) acrosomal integrity. Overall, TYB maintained optimal (P < 0.05) sperm motility compared with Ham's medium, but Ham's medium maintained more (P < 0.05) intact acrosomes than TYB. Slow cooling (0.2 degrees C min(-1)) was optimal (P < 0.05) compared to rapid cooling (1 degrees C min(-1)), and ultra-rapid cooling (9 degrees C min(-1)) was found to be highly detrimental (P < 0.05). Results obtained in TYB with 0% or 4% glycerol were comparable (P > 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling.

267 BIRTH OF DOMESTIC CAT KITTENS OF PREDETERMINED SEX AFTER TRANSFER OF EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERMATOZOA

In cats, sex selection by fertilization of oocytes with sperm sorted into X- andY-chromosome-bearing populations has credible biomedical, commercial, and conservation connotations. Our objectives were (1) to evaluate the efficiency with which embryos could be produced by IVF of in vivo- and in vitro-matured oocytes with cooled sex-sorted sperm after overnight shipment to the sorting facility and overnight return delivery to an IVF laboratory, and (2) to determine if live kittens of predetermined sex (female) could be produced after transfer of embryos derived by IVF of in vivo-matured oocytes with X-chromosome-bearing sperm to recipient females. Semen samples (n = 5) collected from a single male using an artificial vagina were extended in electrolyte-free solution (glucose–BSA) and shipped overnight in an Equitainer (4�C) to the sorting facility. Upon arrival, sperm were stained (9 µm Hoechst 33 342; 75 � 106 sperm mL–1), adjusted to 50 � 106 sperm mL–1 with 4% egg yolk TALP containing 0.002% food dye, and sorted on an SX MoFlo� flow cytometer (Dako, Fort Collins, CO, USA). Purities from resort analysis averaged 94% (X) and 83% (Y). After sorting, sperm were concentrated by centrifugation, suspended in TEST-yolk buffer (Irvine Scientific, Santa Ana, CA, USA), and return-shipped to the IVF lab where they were received 48 h after collection. In vivo-matured oocytes recovered from gonadotropin-treated donors were inseminated in vitro with X-chromosome-bearing sperm. In vitro-matured oocytes obtained from ovaries donated by local clinics were inseminated in vitro with control (cooled/shipped/non-sorted) or X- orY-chromosome-bearing sperm. At 5 h or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006 Theriogenology 66, 59–71). Embryos produced from in vitro-matured oocytes were allowed to develop until Day 8 in a three-step culture system (Pope et al. 2006). Cleavage frequency of in vitro-matured oocytes after insemination with X-, Y-, and control sperm was 33% (40/120), 35% (52/150), and 42% (48/115), and blastocyst development was 50% (11/22), 55% (23/42), and 53% (21/40), respectively (P &gt; 0.05). Incidence of cleavage after insemination of in vivo-matured oocytes with X-sperm was 62% (54/87). On Day 2, 45 embryos (9–16 per recipient) produced by in vitro insemination of in vivo-matured oocytes with X-sperm were transferred by laparoscopy to the oviducts of four Day 1 gonadotropin-treated recipients. Three recipients established pregnancies and delivered litters of one, four, and seven female kittens between Day 62 and Day 66 of gestation. We have demonstrated that sperm-sorting technology can be applied and used effectively in domestic cats and, potentially, should be relevant to the selective breeding of endangered cats.

Criopreservação de sêmen humano: comparação entre métodos de congelação e tipos de envase

PURPOSE: to compare two different methods of freezing and two types of human semen storage during cryopreservation process. METHODS: experimental research in which the cryopreservation of 18 semen samples from 18 volunteers was studied. Following the addition of the cryoprotectant medium, Test-yolk buffer, the semen samples were packaged into 0.25 mL straws or into 2 mL cryotubes and submitted to cryopreservation by slow or rapid methods, in four different treatments: RS (cryopreservation by rapid method and packaged in straws), RT (rapid-cryotubes), SS (slow-straws), and ST (slow-cryotubes). Samples were thawed after 24 hand then maintained at 37oC. Data collected were analyzed by the Student t-test, with p<0.05, using the SPSS computer program for Windows®, version 11.0.0. RESULTS: the motility of spermatozoa decreased after the cryopreservation process. The initial motility rate was 58.1% and motilities after the different methods of cryopreservation were 19.2% (RS), 27% (RT), 21.1% (SS) and 30.3% (ST). There was a significant decrease of the normal morphology. The initial normal morphology was 14.2% and morphologies after the different methods of cryopreservation were 12.8% (RS), 12.6% (RT), 12.6% (SS) and 12.4% (ST). CONCLUSIONS: the slow method of cryopreservation with storage in cryotubes showed the best recovery of motile cells after freezing and thawing. There was no difference among the methods when appraised the morphology.

P-183: A comparative study on TEST-Yolk buffer and human sperm preservation medium on post thaw characteristics of human sperm from pre-freeze specimens

Optimum cryosurvival is dependent not only on freezing methodologies, but also on the type of cryopreservatives used. It seems reasonable to speculate that removing the seminal plasma and selecting highly motile spermatozoa by an optimal sperm processing technique, before freezing, may reduce the loss of viability, motility and acrosome integrity during cryopreservation. Moreover, this method can remove those cells that produce free radicals thereby minimizing peroxidative damage during freeze-thaw procedure.

Effect of cryopreservation on sperm apoptotic deoxyribonucleic acid fragmentation in patients with oligozoospermia

To verify if the increase in sperm apoptotic DNA fragmentation during cryopreservation is greater in oligozoospermic patients than in normozoospermic controls. Controlled prospective study. Patients in an academic research environment. Forty-seven patients with oligozoospermia (concentration <10 x 10(6) sperm/mL) and 30 normozoospermic men. Sperm cryopreservation using a standard protocol with a test-yolk buffer, glycerol as the cryoprotectant. Rate of apoptotic sperm DNA fragmentation as assessed by the terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nickend labeling (TUNEL) assay, graded in apoptotic or nonapoptotic, before and after cryopreservation. An increase in apoptotic DNA fragmentation was observed in all the groups, regardless of sperm concentration. Normozoospermic men presented a smaller rate of apoptotic DNA fragmentation than oligozoospermic patients, both in pre- and postcryopreservation samples. The increase in DNA fragmentation was similar in both groups. Cryopreservation induces apoptotic sperm DNA fragmentation in men, regardless of sperm concentration. Men with oligozoospermia present with higher precryopreservation and postcryopreservation apoptotic sperm DNA fragmentation. The increase in DNA fragmentation is similar in both groups.

Sperm viability in the black-footed ferret ( Mustela nigripes) is influenced by seminal and medium osmolality

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham’s F10 + Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates ( n = 16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen ( n = 5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g, 8 min), resuspended in respective medium, and maintained at 37 °C for 3 h. Sperm motility and forward progression were monitored every 30 min for 3 h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean ± SEM, 513.1 ± 32.6 mOsm; range, 366–791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible ( P < 0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced ( P > 0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more ( P < 0.01) sperm motility than a hyperosmotic Ham’s (700 mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.

O-11: Preliminary Study of the Effect of Test-Yolk Buffer on Human Spermatozoa Prior to ICSI

O-11: Preliminary Study of the Effect of Test-Yolk Buffer on Human Spermatozoa Prior to ICSI

Cryopreservation of spermatozoa from wild-born Namibian cheetahs ( Acinonyx jubatus) and influence of glycerol on cryosurvival

Sperm cryopreservation is a valuable tool for the genetic management of ex situ populations. This study was conducted to assess: (1) semen characteristics of wild-born cheetahs; and (2) the impact of three types of glycerol influence (duration of exposure, temperature, and method of addition) on sperm cryosensitivity. To evaluate the impact of duration of glycerol exposure, spermatozoa were incubated in Test Yolk Buffer (TYB) with 4% glycerol at ambient temperature (∼22 °C) for 15 vs. 60 min before cryopreservation. To evaluate the influence of temperature and method of glycerol addition, spermatozoa were resuspended at ambient temperature either in TYB with 0% glycerol followed by addition of 8% glycerol (1:1 v/v; at ambient temperature vs. 5 °C) or directly in TYB with 4% glycerol. All samples were cryopreserved in straws over liquid nitrogen vapor and evaluated for sperm motility and acrosomal integrity after thawing. Semen samples ( n = 23; n = 13 males) contained a high proportion (78%) of pleiomorphic spermatozoa. Ejaculates also contained a high proportion of acrosome-intact (86%) and motile spermatozoa (78%). Immediately after thawing, a significant proportion of spermatozoa retained intact acrosomes (range, 48–67%) and motility (range, 40–49%). After thawing, incubation in glycerol for 60 min at ambient temperature before freezing decreased ( p < 0.05) sperm motility and acrosomal integrity at one time-point each (pre-centrifugation and post-centrifugation, respectively). However, method or temperature of glycerol addition had no ( p > 0.05) impact on sperm cryosurvival. In summary, (1) wild-born cheetahs produce high proportions of pleiomorphic spermatozoa but with a high proportion of intact acrosomes; and (2) resuspension in 4% glycerol, followed by exposure for up to 60 min at ambient temperature, had minimal effect on sperm motility and acrosomal integrity after cryopreservation. Results indicate the feasibility of cryopreserving cheetah spermatozoa under field conditions, providing a user-friendly method to capture and store gametes to enhance genetic management.

Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus)

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25 degrees C or after slow cooling to 5 degrees C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (+/- s.e.m.) 43.6 +/- 14.2 x 10(6) motile spermatozoa with 33.5 +/- 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P < 0.05) after thawing. All thawed samples fertilised domestic cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5 degrees C resulted in higher (P < 0.05) post-thaw motility and intact acrosomes than glycerol addition at 25 degrees C. In conclusion, good-quality ejaculates can be obtained from Thai fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

115 SPERM CRYOPRESERVATION IN TRAGELAPHINE ANTELOPES

As an integral aspect of our program to develop assisted reproductive technologies in tragelaphine antelopes, we have been evaluating the effects of various extenders on cryosurvival of spermatozoa. Semen was collected by electroejaculation (EEJ) from an eland (Taurotragus oryx, n = 14 EEJs) and a bongo (Tragelaphus euryceros, n = 7 EEJs) male during sedation and standing restraint in a hydraulic chute. Epididymal spermatozoa were collected from the proximal vas deferens, and distal epididymides recovered from four common eland bulls after elective castration at the Hattiesburg Zoo, Mississippi (age = 46 months) and the San Diego Wild Animal Park, California (ages = 18, 20, and 23 months). Testes from a bongo (age = 6.5 yr) and a greater kudu bull (Tragelaphus strepsiceros, age = 7.5 yr) at the Audubon Nature Institute were recovered and processed immediately postmortem. Kudu, bongo, and the Hattiesburg eland testes were transported at ambient temperature whereas the testes from San Diego elands were shipped overnight in a cold-pack container. Sperm processing was done at room temperature (RT = 23°C) in HEPES-buffered Tyrode's solution. Four extenders containing 7% glycerol were evaluated: Biladyl®, TEST Yolk buffer (TYB), Beltsville extender (BF5F), and skim milk. For freezing, sperm samples were initially extended and gradually cooled to 4°C before sequential addition of extender containing glycerol at 4°C; the procedure was then modified by addition of the glycerol fraction at RT before the sample was cooled to 4°C. Spermatozoa were vapor-frozen in 0.5 mL straws placed 5 cm above liquid nitrogen before storage at −196°C. Straws were thawed in a water bath (38°C, 30 s) to evaluate cryosurvival. All eland and bongo electroejaculation procedures produced spermatozoa, although sperm quality varied. Ejaculate volume averaged 5.4 ± 1.2 mL and 3.7 ± 1.1 mL in the bongo and the eland, respectively. Sperm motility at collection, after cooling, and after freezing in the different extenders is presented in Table 1. No immediate decline in sperm motility was observed after adding glycerol to samples, whether at 23°C or 4°C. Bongo spermatozoa lost more motility during freezing than during cooling (15.0 ± 4.4 vs. 5.8 ± 2.0%; P = 0.006); whereas, in eland, motility loss during cooling and freezing was similar (25.3 ± 16.2 vs. 21.1 ± 7.7; P = 0.44). The present results indicate that cooling and freezing tolerance of spermatozoa in tragelaphine antelopes is influenced by species, extender type, and temperature at which a cryoprotectant is added. Table 1. Cryosurvival of ejaculated or epididymal spermatozoa of three antelope species in different extenders

Fecundity of Human Semen Specimens Cryostored and Transported at 5°C Using the Bio-Tranz™ Shipping System

Transport of semen specimens for evaluation and clinical use requires specific protocols for shipment and maintenance of sperm viability during transport. The Bio-Tranz™ shipper is a commercially available system that cools specimens (5°C) during transport. This study was designed to assess the viability and fecundity of semen stored at 5°C for 24hr using the Bio-Tranz™ shipping system. Randomized, prospective study. Semen specimens were assessed for motility and sperm membrane integrity at the time of collection and 24-h after storage in the Bio-Tranz™ shipper (ZDL, Inc., Lexington, KY, USA). In group 1 (n=61), specimens were diluted in test yolk buffer (TYB), processed and used for intrauterine insemination (IUI), leaving an aliquot for storage for 24-h in the Bio-Tranz™. In group 2 (n=67), specimens were diluted in TYB, stored for 24-h in the Bio-Tranz™ and then processed and used for IUI. In both groups, the total motile sperm used for IUI was similar and the females that underwent IUI were standardized for ovulation prediction and time of insemination. The results obtained from this clinical study are summarized in Table 1. The overall sperm characteristics between the fresh specimens (TYB, 0-h) and specimens prepared using TYB and stored via the Bio-Tranz™ shipper for 24-h were within normal limits according to WHO standards. The sperm motility however was reduced in both groups (72.8% vs 57.3%; P<0.05 and 61.8 vs 52.8%; P<0.05). A similar decrease was also noted in the membrane integrity as measured by the hypoosmotic swelling (HOS) test (72.7 vs 61.1; P<0.05 and 65.3 vs 58.7; P<0.05). Furthermore, similar pregnancy rates were obtained between the two patient populations compared (32.8 vs 31.3; P>.05) and this may be due to the fact that the TMS used at IUI were similar for the two groups of patients compared (TMS of 32.6 vs 29.9 x106; P>0.05) respectively. Also, of equal clinical significance is the fact that those pregnancies were achieved within the same time period, which it required 3.3 to 3.5 cycles of IUI’s. Tabled 1 The results obtained show that shipment of semen specimens via the Bio-Tranz™ shipper is possible and can yield similar pregnancy results as fresh semen. Also, the Bio-Tranz™ shipper maintained both adequate sperm viability and fecundity after 24-h of cryostorage. The use of the Bio-Tranz™ shipper is extremely convenient for patients requiring semen evaluation, cryostorage or IUI and other assisted reproductive technologies. The technique is of significant clinical and economic importance to patients and physicians.

Feasibility of Multiple Cycles of Thaw-Refreeze Human Spermatozoa by Standard Vapor Freezing Method

ObjectiveTo evaluate the feasibility of repetead cycles of thaw-refreeze human spermatozoa by using the standard liquid nitrogen vapor freezing method.DesignSemen specimens frozen by standard vapor freezing were thawed at room temperature for 5 minutes and at 37°C for 20 minutes. An aliquot was removed for sperm analysis and the remaining specimen was refrozen by standard vapor freezing without removing the cryomedia (TEST-yolk buffer with glycerol, Irvine, USA). The specimens were stored in liquid nitrogen for 24h and thawed by the same technique. Repeated freeze-thaw cycles were performed until no motile sperm were seen.Materials and methodsSixteen frozen semen samples were obtained from twelve subjects (normozoospermic, n=5; and oligozoospermic, n=7) who required disposal of their samples stored in our sperm bank. Each sample was evaluated for percent total and progressive motility, as well as vitality if no motile sperm was seen, after each cryopreservation cycle. Wilcoxon signed-rank test was used to test for statistical differences.ResultsTabled 1ConclusionsMotile spermatozoa can be obtained after repetead cycles of cryopreservation using the standard vapor freezing method. The sperm ability to resist injury due to the freeze-thawing process is related to the initial semen quality. However, motile sperm obtained after repeated freezing will be only suitable for intracytoplasmic sperm injection. Re-freezing leftover frozen-thawed specimens may be recommended for patients who were not able to freeze multiple specimens, such as cancer patients. Though our results are promising, others studies are required in order to assess the fertility potential of refrozen human semen in cycles of assisted reproduction. ObjectiveTo evaluate the feasibility of repetead cycles of thaw-refreeze human spermatozoa by using the standard liquid nitrogen vapor freezing method. To evaluate the feasibility of repetead cycles of thaw-refreeze human spermatozoa by using the standard liquid nitrogen vapor freezing method. DesignSemen specimens frozen by standard vapor freezing were thawed at room temperature for 5 minutes and at 37°C for 20 minutes. An aliquot was removed for sperm analysis and the remaining specimen was refrozen by standard vapor freezing without removing the cryomedia (TEST-yolk buffer with glycerol, Irvine, USA). The specimens were stored in liquid nitrogen for 24h and thawed by the same technique. Repeated freeze-thaw cycles were performed until no motile sperm were seen. Semen specimens frozen by standard vapor freezing were thawed at room temperature for 5 minutes and at 37°C for 20 minutes. An aliquot was removed for sperm analysis and the remaining specimen was refrozen by standard vapor freezing without removing the cryomedia (TEST-yolk buffer with glycerol, Irvine, USA). The specimens were stored in liquid nitrogen for 24h and thawed by the same technique. Repeated freeze-thaw cycles were performed until no motile sperm were seen. Materials and methodsSixteen frozen semen samples were obtained from twelve subjects (normozoospermic, n=5; and oligozoospermic, n=7) who required disposal of their samples stored in our sperm bank. Each sample was evaluated for percent total and progressive motility, as well as vitality if no motile sperm was seen, after each cryopreservation cycle. Wilcoxon signed-rank test was used to test for statistical differences. Sixteen frozen semen samples were obtained from twelve subjects (normozoospermic, n=5; and oligozoospermic, n=7) who required disposal of their samples stored in our sperm bank. Each sample was evaluated for percent total and progressive motility, as well as vitality if no motile sperm was seen, after each cryopreservation cycle. Wilcoxon signed-rank test was used to test for statistical differences. ResultsTabled 1 ConclusionsMotile spermatozoa can be obtained after repetead cycles of cryopreservation using the standard vapor freezing method. The sperm ability to resist injury due to the freeze-thawing process is related to the initial semen quality. However, motile sperm obtained after repeated freezing will be only suitable for intracytoplasmic sperm injection. Re-freezing leftover frozen-thawed specimens may be recommended for patients who were not able to freeze multiple specimens, such as cancer patients. Though our results are promising, others studies are required in order to assess the fertility potential of refrozen human semen in cycles of assisted reproduction. Motile spermatozoa can be obtained after repetead cycles of cryopreservation using the standard vapor freezing method. The sperm ability to resist injury due to the freeze-thawing process is related to the initial semen quality. However, motile sperm obtained after repeated freezing will be only suitable for intracytoplasmic sperm injection. Re-freezing leftover frozen-thawed specimens may be recommended for patients who were not able to freeze multiple specimens, such as cancer patients. Though our results are promising, others studies are required in order to assess the fertility potential of refrozen human semen in cycles of assisted reproduction.

What is the lowest optimal semen volume for cryopreservation test vials?

Test vials were cryopreserved with semen samples to determine expectations of sperm cryosurvival. Using the lowest volume possible in test vials that accurately represents post-thaw survival of remaining cryopreserved samples provides the greatest volume for future therapeutic use. Studies were conducted to determine the lowest optimal volume of semen to cryopreserve in test vials by evaluating the post-thaw sperm motility and forward progression of semen samples frozen in various aliquots. Additionally, an assessment was made to determine if the liquid nitrogen vapor step in traditional slow-rate sperm freezing can be eliminated when storing samples in a nitrogen vapor freezer. Institutional Review Board approved laboratory study. Ten human semen samples were diluted 1:1 over ten minutes with three aliquots of freezing medium containing TEST Yolk Buffer (TYB) with 12% v/v glycerol (Irvine Scientific, Santa Ana, CA, USA). Samples were then placed into cryovials in duplicate aliquots of 50, 100, 200, 300, 400 and 1000 μl and equilibrated in an ice bath at 4° C for 45 min. One vial of each aliquot volume were placed into liquid nitrogen vapor for 20 min and then into the liquid nitrogen vapor freezer (Method 1). The remaining vials were placed directly into the liquid nitrogen vapor freezer (Method 2). After one week storage specimens were thawed at room temperature. Post-thaw sperm motility and forward progression were determined in duplicate in a blinded fashion. Results were statistically analyzed by ANOVA or Students t-test with independent variables being volume of frozen aliquots and freezing methods used. Post-thaw sperm motility for 50 μl aliquots using Method 1 (27 ± 3; mean ± S.E.) and Method 2 (23 ± 2) were significantly lower than larger aliquots for Method 1 (36 ± 1) and Method 2 (35 ± 1; P<0.005). There was no significant difference in post-thaw sperm motility when comparing 100, 200, 300, 400 and 1000 μl aliquot volumes for Method 1 or 2 (P>0.05). No difference was observed in post-thaw sperm forward progression for Method 1 when comparing various volumes tested. Using Method 2, a significant difference in post-thaw sperm forward progression was noted for 50 μl aliquots (64 ± 2) compared to 300 μl (70 ± 2), 400 μl (72 ± 1), and 1000 μl (72 ± 1; P<0.05). No other significant differences were observed when remaining aliquot volumes were compared. There was no significant difference in post-thaw sperm motility and forward progression when comparing similar volumes of semen using either Method 1 or 2 (P>0.05). When slow-rate freezing semen, test vials should be in a minimum of 100 μl aliquots. Although similar sperm recovery rates can be obtained using larger volumes, the smaller volume allows for more semen allocated to therapeutic use, which may be beneficial to patients providing a limited number of samples. Additionally, after diluting semen samples with freezing medium and cooling to 4° C, samples can be placed directly into the liquid nitrogen vapor freezer without compromising sperm recovery rates.

Preliminary comparisons of a unique freezing technology to traditional cryopreservation methodology of equine spermatozoa

With recent large-breed organization acceptance, the use of frozen semen is gaining more attention in the equine industry. However, cryopreserved stallion semen is commonly associated with poor quality and decreased pregnancy rates as compared with those produced during normal mating or with cooled semen techniques. Therefore our objective was to investigate a new unique freezing technique (UFT) with the intent of improving fertility outcomes. A series of experiments tested the UFT compared with traditional liquid nitrogen methodology in combination with influence of the extenders and stallions used. In Experiment 1, post-thaw motility results of UFT variations were compared with those from liquid nitrogen methods. The averaged post-thaw motility percentages of the 4 UFT treatments were similar when compared with the traditional liquid nitrogen control group ( P = .845). In Experiment 2, 2 egg-yolk–based freezing extenders, Biladyl AB intended for bovine samples and Freezing Medium Test Yolk Buffer used for human samples, were compared. A significant difference in the average post-thaw motilities was found between Biladyl AB (17%) and Freezing Medium Test Yolk Buffer (25%) ( P < .002). In the third experiment, we compared variability among stallions using the UFT with the intention of creating a more consistent outcome. Post-thaw motilities and percent of original motility returns among the 4 stallions were significantly different ( P < .001). In a field trial using shipped semen from a regional stallion station, the UFT demonstrated very promising results. In conclusion, the UFT may potentially be used as an alterative freezing method to replace current liquid nitrogen methodology. However, further investigation is needed to refine techniques.

The effect of a two-hour, room temperature incubation of human spermatozoa in TEST-yolk buffer on the rate of fertilization in vitro.

To reassess the use of TEST-yolk buffer (TYB) in an in vitro fertilization (IVF) program by comparing fertilization rates achieved in a glucose-free cleavage medium by the standard IVF preparation of sperm versus a 2-h, room temperature incubation of sperm in TYB. Oocytes collected for IVF were randomly split into two groups and inseminated with either TYB-treated sperm or IVF-prepared sperm. Stanford Reproductive Endocrinology and Infertility Center. Fifty couples undergoing IVF with at least 10 mature oocytes. Fertilization rates in vitro. Fertilization rates were significantly higher (p = 0.015) with TYB treatment. The average 2PN fertilization rate was 49.6% (188/379) for the IVF group and 57.4% (221/385) in the IVF with TYB group. A 2-h, room temperature incubation of sperm in TYB produces significantly higher 2PN fertilization rates as compared to standard IVF preparation of sperm in a current generation cleavage medium.

212EFFECT OF CRYOPRESERVATION METHODS AND PRE-CRYOPRESERVATION STORAGE ON BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATES) SPERMATOZOA

In conjunction with artificial insemination (AI) and sperm preservation, sperm sexing technology has great potential as a population management strategy for captive bottlenose dolphins. Successful AI using fresh spermatozoa (Robeck TR et al. 2001 CRC Marine Mammal Medicine 193–226) and flow cytometric analysis of bottlenose dolphin spermatozoa (Garner DL and Seidel GE Jr 2002 CSAS Symposium 2–13) support this approach. For sperm sexing, methods for short-term storage of semen in a liquid state are required to enable transport of spermatozoa to the sorting laboratory. In addition, cryopreservation techniques must be optimized for long-term storage of sexed spermatozoa. Our objectives were to assess: (i) 3 cryopreservation methods×2 straw sizes×3 thawing rates (Exp. 1) and (ii) effects of liquid storage for 24h (pre-cryopreservation) and sperm concentration at freezing (Exp. 2) on post-thaw characteristics (PT) of bottlenose dolphin spermatozoa. For Exp. 1 and 2, 4 ejaculates (collected by manual stimulation)×3 males (aged 14–34yr)×4 replicates were used. For Exp. 1, semen was frozen in 0.25-mL (SM) and 0.5-mL straws (LG) by 3 methods (Mt) (Mt1: lactose, egg yolk, −32°Cmin−1; Mt2: lactose, egg yolk, 1.5% Equex STM (Nova Chemical, Calgary, Canada), −19.7°Cmin−1; Mt3: Test yolk buffer (TYB), −116°Cmin−1). All Mt had 3% glycerol. Samples were thawed using a slow (S: 2.8°Cs−1), medium (M: 8.8°Cs−1) or fast (F: 21°Cs−1) rate. In Exp. 2, ejaculates were divided into 4 aliquots for dilution (1:1) and stored at 4°C with EquiPro® (EP4°C, Minitube, Verona, WI, USA) and TYB (TYB4°C) or at 21°C with Androhep EnduraguardTM (AH21°C, Minitube) or no dilution (NEAT21°C). After 24h, samples were frozen and thawed using Mt3×SM×F at 10×106 spermmL−1 (LOW) or 100×106 spermmL−1 (STD). PT evaluations of motility (total motility [TM], % progressive motility [PPM], kinetic rating [KR, 0 to 5]) and acrosomal status (Spermac® , Minitube) were performed at 30min and 6h after dilution (1:1) with AH at 21°C. For statistical analysis (ANOVA), a sperm motility index (SMI=TM×PPM×KR) was calculated and expressed as % of initial SMI. For all ejaculates, initial TM and PPM were greater than 85% and KR was 5. In Exp. 1, at 6h PT, %SMI was highest for Mt3×LG×M (45.5±8.7) and Mt3×SM×F (44.8±11.9). For Exp. 2, %SMI at 0h PT was higher for samples stored at 4°C than at 21°C (TYB4°C 41.0±8.4, EP4°C: 36.7±7.7, NEAT21°C: 23.8±8.6, AH21°C: 14.8±8.6, P&amp;lt;0.001) and, with the exception of AH21°C, was similar between the LOW and STD concentration. At 6h PT, %SMI for all treatments was higher for STD than LOW concentration (P&amp;lt;0.05). Acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol maintained high levels of the initial characteristics of ejaculated spermatozoa. Transport of semen for sex pre-selection and cryopreservation within 24h may be feasible, but impact of storage time on functional capacity of dolphin spermatozoa is unknown.

202FERTILIZING ABILITY OF EPIDIDYMAL CAT SPERMATOZOA AFTER CRYOPRESERVATION OR STORAGE AT 4°C.

The possibility of harvesting cat epididymal spermatozoa from excised testis represents a potentially important tool for preserving valuable genetic material from males that die unexpectedly. The purpose of this study was to evaluate plasma membrane integrity, acrosomal status and in vitro fertilizing capability of epididymal cat sperm after short- and long-term storage at 4°C and after cryopreservation. Spermatozoa were collected by flushing the cauda epididymis and the vasa deferentia removed from adult cats undergoing orchiectomy. Spermatozoa were split into three aliquots: A-fresh, B-frozen, and C-cooled. The A portion was immediately tested on the day of collection (control), the B portion was frozen in pellets in Test Yolk Buffer (TYB) with 8% glycerol, while the C portion was extended in TYB and stored at 4°C for 1 day, 2 days or 7 days. Cat oocytes recovered from minced ovaries were matured for 24h in TCM 199+1UI/mL hCG+0.5UI/mL FSH+0.3% BSA at 38.5°C in 5% CO2. In vitro fertilization (IVF) of in vitro-matured (IVM) oocytes was performed using spermatozoa from portions A, B or C in modified Tyrode’s solution supplemented with 0.6% BSA at 38.5°C in 5% in air. Before IVF, aliquots of sperm samples from the three portions were stained simultaneously with fluorescein isothiocyanate-labelled Pisum Sativum agglutinin (FITC-PSA) and propidium iodide to evaluate the percentage of plasma membrane-intact spermatozoa with acrosomes present. At least 200 spermatozoa were counted in duplicate for each sample. After 24h of incubation, to evaluate fertilization rate, the oocytes were stained with aceto-lacmoid. Oocytes with two pronuclei, presumably male and female, were classified as fertilized. The percentages of spermatozoa maintaining plasma membrane integrity and intact acrosomes after 1 day or 2 days of storage at 4°C were 75% and 65%, respectively, and were similar to that of fresh epididymal spermatozoa (78%). However, the percentages of spermatozoa with intact plasma membranes that had intact acrosomes after 7 days of storage at 4°C (52%) or after cryopreservation (48%) were significantly lower (P&amp;lt;0.01, ANOVA) than those of samples stored for 1 or 2 days at 4°C. As shown in the Table, fertilization rates of oocytes inseminated with fresh spermatozoa and spermatozoa stored for 1 or 2 days were similar. In contrast, the fertilization rates of oocytes inseminated with spermatozoa that had been cryopreserved or stored for 7 days were significantly lower than those obtained with spermatozoa used immediately after collection or storage for 1 day. From our results, we suggest that storage of epididymal spermatozoa at 4°C may be a potentially useful method for temporary storage of spermatozoa from endangered cats, and may allow more efficient use and long-distance transport of genetically important germplasm. This work was supported by MIUR (ex 40%). Table 1 In vitro fertilization of in vitro-matured cat oocytes with fresh, stored at 4°C and cryopreserved epididymal cat spermatozoa

Single sperm cryopreservation on cryoloops: an alternative to hamster zona for freezing individual spermatozoa

Methodology enabling cryopreservation of individual spermatozoa in extreme cases of oligozoospermia would tremendously benefit patients. This study explores the use of a nylon loop for cryopreservation of small quantities of spermatozoa, and also describes a novel technique for freezing individual spermatozoa with this cryoloop. Experiments were conducted to compare sperm recovery and viability after cryopreservation in conventional vials versus on the cryoloops. Discarded human sperm specimens with varying parameters were utilized. The study also examines two different glycerol-based cryoprotectants, with and without test yolk buffer. For single sperm cryopreservation, 5–10 spermatozoa were selected and loaded onto cryoloops with the aid of a microscope and micromanipulation equipment. Sperm function testing was performed on both human and bovine spermatozoa frozen on cryoloops. Microquantities of spermatozoa frozen on cryoloops exhibited overall motility and viability parameters similar to control samples frozen in cryovials. Individually selected spermatozoa cryopreserved on loops were easily recovered and post-thaw motility was generally good. Sperm function testing demonstrated that both human and bovine spermatozoa cryopreserved on loops were able to undergo sperm head decondensation when injected into oocytes. Cryoloops may be an excellent alternative to hamster zonae for cryopreserving small numbers of human spermatozoa.