Pre-freeze Motility Research Articles

#83 : Human Sperm Cryo-Survival Rate

Background and Aims: Sperm cryopreservation plays an important role in the suite of services available to fertility patients. The ability to preserve the motility and viability of human spermatozoa is a key component in delivering successful assisted reproductive technologies (ART) outcomes. Cryopreservation methods expose sperm to stress, resulting in cellular damage which consequently leads to a reduction in their survival rate. The aim of this study was to evaluate the average cryo-survival rate of semen samples frozen in a clinical setting, to help improve and manage patient expectations prior to accessing this service. Method: Pre and post sperm freeze data from patients who presented at the Genea Sydney clinic for sperm cryopreservation between 2017 and 2022 was analysed. The post-thaw survival rate for 1467 specimens was assessed by averaging the total post-thaw motility with total pre-freeze motility. Only ejaculated samples, frozen on-site, who had a raw motility calculated as well as a post-thaw motility analysis able to be performed were included in the study. Results: The range of post-thaw survival was 57% to 67% per annum over the six years of examined data, with an overall cryo-survival rate of 62%. Conclusions: The data shows a consistent recovery rate of post-thaw sperm motility over the time period analysed. Literature indicates average sperm cryo-survival rates range from 30% to 50%, and previous advice to patients has been to expect around 50% sperm survival. With this new data, Genea can revise its guidance to match the current average figure of around 60%. In addition, future internal freeze optimisation studies can use this figure as a benchmark to improve upon techniques and processes.

The effect of gonadal and extragonadal malignant cancers on sperm quality.

BackgroundMany studies have reported that testicular germ cell tumours (TGCTs) give rise to a decrease in sperm production and may further lead to infertility. However, little is known regarding sperm production in extragonadal germ cell tumours (EGCTs). We have studied the differences in the characteristics and sperm quality of patients with testicular cancer and patients with EGCTs.MethodsWe collected and analysed demographic factors, semen parameters, and the disposition of banked sperm.ResultsTGCTs accounted for the majority of malignant cancers compared with EGCTs (83.9% vs. 16.1%, respectively). There were no statistically significant differences in semen volume, pre-freeze concentration, pre-freeze motility, post-thaw concentration, post-thaw motility or recovery rate between patients with TGCTs and those with EGCTs. Both patients with TGCTs and EGCTs showed low pre-freeze and post-thaw sperm concentrations. Men with a seminoma had a higher median level of thaw-sperm motility in their ejaculate than men with non-seminomas. TGCT (67%) and EGCT (72%) patients chose to keep their specimens in storage.ConclusionsOur study provides evidence that both patients with TGCTs and EGCTs have a high risk for impaired semen quality and gonadal dysfunction. This suggests that oncologists and patients should consider the effects of both TGCTs and EGCTs on fertility among males.

MP31-02 MICROTESE AND CRYOPRESERVED SPERM - A DECISION-MAKING DILEMMA FOR PATIENTS AND PROVIDERS

You have accessJournal of UrologyInfertility: Therapy (MP31)1 Sep 2021MP31-02 MICROTESE AND CRYOPRESERVED SPERM - A DECISION-MAKING DILEMMA FOR PATIENTS AND PROVIDERS Johnathan Doolittle, Darren Bryk, Scott Lundy, and Sarah Vij Johnathan DoolittleJohnathan Doolittle More articles by this author , Darren BrykDarren Bryk More articles by this author , Scott LundyScott Lundy More articles by this author , and Sarah VijSarah Vij More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002035.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: A known disadvantage of frozen microdissection testicular sperm extraction (mTESE) is the negative impact of cryopreservation and subsequent thawing of any sperm that are obtained. A post thaw (PT) sample provides insight in to how the sample withstands this stress and the predicts likelihood of sperm available for intracytoplasmic sperm injection (ICSI). If a PT cannot be performed due to such low numbers of sperm identified or no pre-freeze motility, providers and couples arrive at a challenging decision in their fertility pathway - pay for an additional sperm retrieval procedure or proceed with a sample that may not be sufficient for ICSI, requiring a back-up plan. Our study sought to characterize the ICSI outcomes after retrieval of motile sperm during frozen mTESE at our institution. METHODS: A retrospective chart review was conducted from 2012 through 2020 to identify all men that underwent frozen mTESE with successful retrieval of motile sperm. Prior sperm extraction or biopsy, testis size, hormone levels, genetics and prior semen analyses were recorded. A PT and recommendations regarding ISCI were made by the lead embryologist at our institution. When available, ISCI outcomes were recorded. RESULTS: Of the 88 men who underwent frozen mTESE, motile sperm were identified in 26.1% (23/88). 16 of these samples were assessed with a PT, of which 12/16 had PT motility. Of these 12, 10 specimens were deemed sufficient to proceed to ISCI by the embryologist whereas the remaining 2 couples were advised to consider fresh mTESE. ICSI records were available for 7 of the 12 patients with PT motility. All 7 cycles led to embryos for transfer, resulting in 6 pregnancies and 5 live births. Of the 11 men without PT motility, ICSI records were available for 6 couples. 3 underwent subsequent fresh mTESE all leading to embryos for transfer but no pregnancies, and 3 proceeded straight to ICSI, 2 of which created an embryo for transfer, with 1 resulting in an ongoing pregnancy. CONCLUSIONS: Of those men who have successful frozen mTESE, as defined as recovery of motile sperm, some may be advised to pursue fresh mTESE based on poor PT parameters or absence of PT due to poor pre-freeze parameters. This creates a very difficult decision for couples. Although frozen mTESE potentially spares the female partner an unnecessary IVF cycle, we often place couples in a difficult position to make a decision about whether to proceed to ICSI absent PT or in the setting of poor PT motility. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e555-e555 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Johnathan Doolittle More articles by this author Darren Bryk More articles by this author Scott Lundy More articles by this author Sarah Vij More articles by this author Expand All Advertisement Loading ...

PSV-5 Effect of Breed and Barn Location on Semen Quality at an Artificial Insemination Facility

Abstract A better understanding of the factors that influence collection and semen quality for AI sires is needed. Our objectives were to evaluate the effect of breed and environmental factors on semen characteristics from 49,086 ejaculates analyzed by a single technician at a beef semen collection facility. Bulls were housed in one of 5 barns based on health testing and purpose of semen collection. Multiple ejaculate characteristics were evaluated using regression models; fixed effects included breed, age, and barn. Ejaculate volume differed (P < 0.001) between Angus (8.5 ml) and Horned Hereford (7.5 ml). Concentration of spermatozoa was lower (P < 0.001) for Angus compared with Simmental, Charolais, and Horned Hereford (1009, 1100, 1252, and 1197 x 106/ml, respectively). Horned Hereford had greater (P < 0.01) concentration of spermatozoa compared with Red Angus (1197 and 1046 x 106/ml, respectively). Simmental had greater (P < 0.01) pre-freeze motility when compared with Angus, Red Angus, and Horned Hereford (54, 50, 49 and 49%, respectively). Average production of 0.5 CC frozen straws per collection was greater (P < 0.001) for Simmental (244) when compared with Red Angus (205) and Angus (223). Pre-freeze motility (% motile) varied from 48% to 53% between the five barns (P < 0.001). The average concentration of spermatozoa varied from 1000 (x 106/ml) to 1155 (x 106/ml), with Barn 1 producing the lowest per ejaculate (P < 0.001). Bulls in Barn 1 produced a greater amount (P < 0.001) of primary abnormalities than Barn 4 and more (P < 0.001) secondary abnormalities than Barn 3. Overall, Simmental, when compared with Angus bulls, produced the greatest number of straws per collection, with the highest pre-freeze motility. Recognizing breed differences and the effect of barn location may help improve overall efficiency, and producer expectations of semen collection at this facility.

Influence of non-genetic factors on semen characteristics of Malabari bucks

A study was designed to analyse the effect of non-genetic factors on semen quality traits of Malabari bucks maintained at Frozen Semen Bank, Madras Veterinary College, Chennai, Tamil Nadu. Data on 675 ejaculates collected from four Malabari bucks spread over six years were used. The overall least-squares means for semen volume, sperm concentration, mass activity, initial sperm motility, pre-freeze motility, post-thaw motility and number of frozen semen doses per ejaculate were 0.81±0.01 ml, 3144±17.24 million per ml, 4.80±0.02, 79.60±0.00%, 76.31±0.00%, 35.16±0.00% and 27.16±0.59 doses, respectively. The bucks had significant influences (P<0.01) on all the traits. Season had highly significant influence on semen volume, sperm concentration, initial sperm motility, post-thaw motility, number of doses per ejaculate (P<0.01) and pre-freeze motility (P<0.05). Winter season was found to have the highest semen volume, post-thaw motility and number of doses per ejaculate, whereas initial sperm motility was highest in summer and sperm concentration and pre-freeze motility in south-west monsoon. The effect of ejaculate number was significant on post-thaw motility (P<0.01), sperm concentration and pre-freeze motility (P<0.05). Among ejaculates, significantly higher sperm concentration, pre-freeze and post-thaw motility were observed in second ejaculates. The semen samples obtained during winter season and in second ejaculate were superior in seminal characters and more suitable for cryopreservation.

Seasonal Variation and Age-Related Changes in Semen Quality of Limousin Bull in Indonesian Artificial Insemination Center

The objective of this study was to determine the effect of season and age on semen quality (individual motility, mass motility, concentration, volume, pre-freeze motility, and post-thaw motility) of Limousin Bull under the tropical condition in Indonesian artificial insemination center. This study used a large data set recorded over eight years (2011 until 2018) of production period (n = 6758 ejaculates, n = 19 bulls) retrieved from Singosari National Artificial Insemination Center. Rainfall and other climatic data were collected from Malang Meteorological, Climatology, and Geophysics Agency, Indonesia. Seasonal effect was grouped into three groups, i.e. rainy season (total rainfall per month &gt;200mm), humid season (total rainfall per month = 100-200 mm), and dry season (total rainfall per month &lt;100). Bull age is grouped into seven groups (2, 3, 4, 5, 6, 7 and 8). Data were analyzed using Mixed Model procedure classifying effect of season and age on fixed effect and bull as a random effect. Season had a highly significant effect on individual motility, pre-freeze motility, and post-thaw motility (P&lt;0.001), and did not have a significant effect on concentration, volume, pH, and mass motility. Individual motility, pre-freeze motility, and post-thaw motility were highest in the rainy season and declined in the dry season. Bull age had a highly significant effect on individual motility, concentration, volume, pre-freeze motility, and post-thaw motility (P&lt;0,001). Bull age did not have a significant effect on mass motility and pH. Two- and three-years old bulls have superior individual motility and concentration. However, it is decreasing at age three until eight years old. The lowest volume was at age two and increased gradually until age eight. Meanwhile, pre-freeze motility and post-thaw motility increased at two to five years old and then declined until eight years old.

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.

Validation-verification of a highly effective, practical human testicular tissue in vitro culture-cryopreservation procedure aimed to optimize pre-freeze and post-thaw motility.

Purpose The aim of our paper was to validate a testicular biopsy procedure that simplifies handling, processing, and cryopreservation, while at the same time optimizes sperm motility before freezing and after thawing.MethodsTwo prospective studies were conducted to verify, optimize, and understand the virtues of pre-freeze testicular tissue IVC at different temperatures (21, 30, or 37 °C). Testicular tissue was obtained from clinical specimens designated for whole tissue cryopreservation (i.e., intact mass of tubules) and/or for fresh use in IVF-ICSI cycles. Whole testicular biopsy pieces (1–3 mm3) were diluted in glycerol containing freeze solutions, slow cooled to 4 °C and then rapidly frozen in LN2 vapor. Fresh and post-thaw testicular biopsy tissue were evaluated for changes in the quantity (%) and pattern of motility (I–IV: twitching to rapid progression, respectively) over a 1 week duration. The clinical effectiveness of IVC-cryopreserved whole testicular biopsy tissue was also validated analyzing fresh embryo transfers.ResultsMore reliable recovery of motile testicular sperm was achieved using whole tissue freeze preservation combined with IVC (24–96 h) post-acquisition at an incubation temperature of 30 °C compared to ambient temperature (21 °C) or 37 °C. Up to 85 % of the pre-freeze motility was conserved post-thaw (+3 h) for easy ICSI selection. Sperm longevity was optimized to fresh tissue levels by implementing testicular biopsy sucrose dilution post-thaw. Favorable clinical outcomes were proven using frozen-thawed testicular biopsy sperm for ICSI.ConclusionsBy employing minimal tissue manipulation, integrating pre-freeze IVC processing at 30 °C and the freezing of whole testicular biopsy tissue, we have reduced the labor and improved the efficacy of processing testicular tissue for freeze-preservation and subsequent ICSI use.

Factors impacting the success of post-mortem sperm rescue in the rhinoceros

The goal of this study was to identify factors that influenced the ability to successfully rescue sperm post-mortem from rhinoceroses maintained in North American zoos. Factors considered included procedural technicalities, individual rhinoceros characteristics and timing. Gross testicular pathology was noted in 17.4% of males (4/23) but did not impact sperm recovery except in one case of azoospermia (4.3%). Of the males in which sperm recovery was attempted (n=21), 62% yielded quality samples considered adequate for cryopreservation (≥30% motility with ≥2.0 forward progressive status). A high percentage of males (70.6%; 12/17) from which reproductive tissue was removed and cooled ≤4h after death yielded quality sperm samples, whereas only 25% (1/4) of males from which tissue was removed>4h after death yielded quality samples. Quality samples were recovered 1–51h post-mortem from rhinoceroses 8 to 36 years old. Neither type of illness (prolonged or acute), or method of death (euthanasia or natural) affected the ability to harvest quality samples (P>0.05). The Indian rhinoceros yielded significantly more sperm on average (40×109) than the African black rhinoceros (3.6×109; P<0.01) and the African white rhinoceros (3.2×109; P<0.05). Across all species and samples assessed (n=11), mean post-thaw sperm motility (41%), was only 15% less than pre-freeze motility (56%) and only decreased to 22% during the 6h post-thaw assessment period. Rhinoceros sperm rescue post-mortem is relatively successful across a wide range of variables, especially when tissues are removed and cooled promptly after death, and should be considered standard practice among zoos.

71 EFFECT OF CHOLESTEROL-LOADED CYCLODEXTRINS ON PRE- AND POST-THAW FUNCTION OF FELID SPERM

Propagation of genetically diverse felid populations would benefit from more effective assisted reproduction strategies, including enhanced methods for sperm cryopreservation. In felids, sperm cryopreservation has been improved by substituting soy-lecithin for egg yolk in cryomedium (Vick et al. 2012 Theriogenology 78, 2120–2128). In other species, such as elephants (Kiso et al. 2012 Reprod., Fert. Dev. 24, 1134–1142) and cattle (Purdy et al. 2004 Cryobiology 48, 36–45), the addition of cholesterol-loaded cyclodextrins (CLC) to sperm before freezing has been shown to produce superior cryopreservation results. In this study, our objectives were to (1) assess cholesterol content of cat sperm membranes and capacitation status following incubation with CLC; (2) evaluate post-thaw sperm motility, acrosome status, and fertility in vitro following CLC treatment and freezing in a soy-based cryomedium; and (3) conduct a preliminary assessment of cholesterol content in nondomestic cat sperm. Freshly collected domestic cat sperm (n = 2 males, 3–4 ejaculates/male) were incubated with CLC (0, 1.5, or 3.0 mg mL–1), and cholesterol levels were measured using an Amplex Red Cholesterol Assay. Sperm aliquots from each CLC concentration were treated with calcium ionophore (2 μM, 30 min) during in vitro incubation and stained with fluorescein isothiocyanate/PNA to evaluate induced acrosomal loss. To assess post-thaw parameters, cat sperm treated with CLC were frozen in straws using soy-lecithin cryomedium, thawed, and cultured in vitro over time. To evaluate fertility, oocytes were collected laparoscopically from gonadotropin-treated domestic cats (n = 7 females, 147 oocytes total) and inseminated with low numbers of thawed-frozen sperm pretreated with 0 or 1.5 mg mL–1 CLC. Data were analysed using ANOVA and mean differences assessed with Fisher l.s.d. or chi-squared analysis. Sperm cholesterol levels were increased (P &lt; 0.05) after exposure to both 1.5 and 3.0 mg mL–1 CLC. Prefreeze motility was decreased (P &lt; 0.05) and capacitation was delayed at 3.0 mg mL–1 CLC relative to treatment with 0 or 1.5 mg mL–1 CLC. Both post-thaw motility and percentage of acrosome intact sperm were reduced (P &lt; 0.05) with the highest CLC concentration, but results were similar (P &gt; 0.05) for 0 and 1.5 mg mL–1 CLC. Fertilization percentages did not differ (P &gt; 0.05) between treatment groups (0 CLC, 33.3%, 25/75; 1.5 mg mL–1 CLC, 26.4%, 19/72). Preliminary results from a single cheetah (Acinonyx jubatus) and single fishing cat (Prionailurus viverrinus) suggest that sperm membrane cholesterol may be lower compared to the domestic cat. Cholesterol content appeared to increase in both species after exposure to 1.5 mg mL–1 CLC. In summary, our findings suggest CLC treatment increased cholesterol content of felid sperm membranes. The higher CLC concentration was detrimental to sperm motility, capacitation, and post-thaw sperm traits. The lower CLC concentration did not improve post-thaw sperm function in domestic cats.Research supported by the Procter &amp; Gamble Wildlife Conservation Scholarship Program.

CRYOPRESERVATION OF RED BROCKET DEER SEMEN (MAZAMA AMERICANA): COMPARISON BETWEEN THREE EXTENDERS

In recent years, genome banks have grown as a way of maintaining the genetic variability of populations. However, the quality of gamete cryopreservation will determine their efficiency. This study aimed to evaluate prefreeze and postthaw sperm motility, vigor, membrane integrity, and morphology of semen of red brocket deer (Mazama americana) using three extenders: E1, Tris-Yolk; E2, Tes-Tris-Yolk; and E3, Tes-Tris-Yolk-Equex. Six bucks were used, and three collections per buck were performed at 90-day intervals. Before freezing, semen volume, ejaculate concentration, motility, vigor, membrane integrity, and sperm morphology were evaluated. To compare the effect exerted by the extenders after sample thawing, further analyses of sperm motility, vigor, membrane integrity, and morphology were performed. Mean ejaculate volume and sperm concentration were 365.33 +/- 120.5 microL and 2,675.73 +/- 810.4 sperm/mL, respectively. Prefreeze motility for the extenders showed no significant differences (approximately 60%). Postthaw motility (E1 = 16.33 +/- 5.5, E2 = 5.44 +/- 5.2, E3 = 24.66 +/- 10.0) was significantly different between E2 and E3, whereas postthaw vigor (E1 =2.66 +/- 0.8, E2= 1.89 +/- 1.2, E3 = 3.83 +/- 0.4) was greater for E3 (P < or = 0.05). Analysis of postthaw membrane integrity revealed no significant differences between the extenders regarding counts of cells presenting intact membranes; however, E3 promoted the lowest number of cells with damaged membranes and higher cell counts for partially damaged membranes (P < or = 0.05). Analysis of sperm morphology revealed an increase in severe abnormalities when using E2 and E3 (P < 0.05). However, observation verified that counts of altered cells were lower using E3 than E2, suggesting a protective effect of Equex. These findings indicate that E3 promoted better semen quality postthaw. However, the performance of this extender in protecting sperm cells of M. americana during freezing was lower than that verified for other species. Thus, further studies are needed for characterization of red brocket deer semen and the optimization of extender for sperm cryopreservation.

Effect of pre-freeze semen quality, extender and cryoprotectant on the post-thaw quality of Asian elephant (Elephas maximus indicus) semen

Semen cryopreservation and artificial insemination (AI) are potentially valuable methods for supporting the breeding management of endangered species like the Asian elephant. Cryopreservation of Asian elephant semen has however proven problematic with respect to maintenance of both adequate semen quality and fertility post-thaw. In this study, nine ejaculates from three adult bulls were used to compare the influence of extender (TEST versus INRA96®) and penetrating cryoprotectants (3% glycerol, 5% glycerol and 4% methylformamide) on post-thaw semen quality. We demonstrate that not only the freezing process, but also the quality of the semen before freezing, significantly influences the freezability of Asian elephant semen. Pre-freeze motility, viability, semen volume, semen pH, sperm concentration and the incidence of sperm mid-piece and tail abnormalities all significantly (p<0.05) affected post-thaw semen quality. While extender and cryoprotectant did not significantly affect any of the above semen quality parameters post-thaw, the skim-milk based extender (INRA96®) preserved DNA integrity better (p<0.05) than the egg yolk extender (TEST). Considerable between-ejaculate variation in all post-thaw semen quality parameters was also noted. It is concluded that strict criteria for semen quality is essential for the selection of Asian elephant bull ejaculates suitable for cryopreservation; stricter initial selection should improve the mean post-thaw quality.

Cryopreservation of epididymal sperm collected postmortem in the Tasmanian devil (Sarcophilus harrisii)

Effective sperm cryopreservation protocols are limited to a small number of marsupial species. In this study, postmortem gamete rescue (PMGR) epididymal sperm samples from Tasmanian devils (N = 34) euthanized due to the fatal Devil Facial Tumor Disease were used to develop long-term sperm storage techniques for the species. Cryoprotectant toxicity associated with equilibration of sperm samples in a TEST yolk diluent (TEST; 189 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 85 mM Trizma base [Tris], 11 mM glucose, 20% vol/vol egg yolk; pH 7.1, and 315.0 ± 5.0 mOsm/kg) with a final concentration of 0.06 M trehalose, or 4%, 10%, and 18% vol/vol of either glycerol or dimethyl sulfoxide (DMSO), was examined over 12 h at 15 °C. Trehalose supplementation resulted in an immediate decline (P < 0.05) of total motility. After 12 h, total motility was reduced (P < 0.05) in treatments containing 18% glycerol, and 10% and 18% dimethyl sulfoxide. The effects of final glycerol concentration (4% and 10%), glycerol equilibration duration (10 min 1 h, or 3 h) prefreeze, freezing rate and the addition of 0.10 M lactose or a combination of 0.10 M lactose and 0.11 M raffinose were assessed during three experiments on the cryopreservation of postmortem gamete rescue samples in TEST. In all experiments, motility and viability were reduced (P < 0.01 postthaw). Samples cryopreserved in TEST supplemented with lactose or lactose with raffinose using a fast freezing rate (−8 °C/min from 4 to −40 °C, then −65 °C/min until −165 °C) produced the highest (P < 0.05) postthaw motility (18.6 ± 5.5% and 16.9 ± 8.5%, respectively), which represented 35% to 48% retention of prefreeze motility. These results apparently were the best postthaw results of dasyurid sperm reported to date and will help lay the foundations for developing assisted reproductive technologies for marsupial species.

Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing

The objective of this study was to evaluate the effects of two commercially available semen extenders on the motility of cryopreserved goat sperm and to simplify the cryopreservation protocol. Individual goat ejaculates were split and processed in parallel for freezing in either commercially available soy-based extender (Bioxcell®) or egg yolk-based extender (Irvine TYB). Sperm quality was assessed using total and progressive sperm motility, measured by computer-assisted sperm analysis (CASA). Total motility was higher for samples processed in soy-based extender, both at pre-freeze (P = 0.002) and at post-thaw (P < 0.0001). Progressive motility was higher for semen processed in soy extender at post-thaw (P < 0.0001). Approximately 10% of samples processed in egg yolk-based extender had a large (> 50%) reduction in total motility prior to freezing. However, this type of extreme reduction in pre-freeze motility did not occur in semen samples processed in soy extender. In addition, the use of soy-based extender eliminated the need for a time-consuming sperm washing protocol. We concluded that a commercially available soy-based extender was superior to an egg yolk-based extender in preserving motility of cryopreserved goat sperm, using a two-step method.

Sperm quality assessments for endangered razorback suckers Xyrauchen texanus

Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckers Xyrauchen texanus collected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167-343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (s.e.m.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2; P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2; P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23-82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985; P<0.0001). Count (FCM) was negatively correlated with post-thaw viability (r=-0.83; P=0.0116) and mitochondrial function (r=-0.91; P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=-0.77; P<0.0001) and pre-freeze viabilities (r=-0.66; P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.

112 EFFECT OF EGG YOLK CONCENTRATION IN TEST BUFFERED EXTENDER ON SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION

Our purpose was to examine the effect of egg yolk concentration (EY; 2, 5, or 10%) on in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST-buffered extender (TYB). Testes were transported in HEPES saline; epididymes were dissected in HEPES 199 medium (He199) and repeatedly sliced. The sperm suspension was filtered (40 μ), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 g for 20 min. Aliquots of the sperm pellet were extended in TYB containing 2, 5, or 10% EY. After cooling to 4°C, samples were diluted 1:1 with TYB containing 2, 5, or 10% EY + 12% glycerol in 4 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (-80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (˜22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122 centrifuged at 200g for 10 min, and pellets resuspended in He199. Motility (Mot, phase contrast, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at 0 h, after gradual cooling to 4°C and after freezing at 0 and 3 h post-thaw (37°C). Ten replicates were done. Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in TYB + 2% egg yolk or HSPM (no egg yolk) in droplets (1 million sperm/mL) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured until blastocyst development was evaluated (Day 8). There were no treatment differences at any time or temperature point for the 3 sperm characteristics evaluated (one-way ANOVA; P &gt; 0.05). As shown in the Table 1, at 0 h post-thawing, sperm in each group retained ˜70% of their initial pre-freeze motility. After 3 h of post-thaw incubation, motility decreased to ˜50% of the pre-freeze value. Cooling to 4°C did not affect membrane integrity or acrosomal status, but post-thaw values were reduced by 30-35% as compared with pre-freeze. Cleavage frequency and blastocyst development of 284 IVM oocytes after IVF using sperm frozen in TYB + 2% EY and HSPM were 53 v. 52% and 42 v. 38%, respectively (P &gt; 0.05). In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in a cryoprotectant solution containing minimal egg yolk (2%). Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage

Conception rate according to semen characteristics in a series of 15 364 insemination cycles: results of a multivariate analysis.

The study was undertaken on 1438 ejaculates from 342 donors. The sperm count, volume, prefreeze and postthaw motility were evaluated for each ejaculate and the morphology for the first ejaculate of each donor. The success rate was found to increase steadily with the sperm count, prefreeze and postthaw motility. For the volume and percentage of all abnormal forms, the success rates are stable up to 5,5 ml and 40% respectively and then decrease. Among all the abnormal forms considered, only the microcephalics and irregular heads were found to be linked to the success rate. As most of the studied characteristics are correlated, a multiple stepwise regression was completed in order to determine the proper role of each variable. The most predictive variable is the postthaw motility. The second variable is the percentage of microcephalics. The other variables bring no further information. In particular, at a given postthaw motility, the prefreeze motility and consequently, the loss in motility have no influence on the success rate.

Between and within subject correlations and variances for certain semen characteristics in fertile men.

Between subject and within subject variances and correlations for different semen characteristics have been determined for a population of 315 fertile men who provided one or more ejaculates within an abstinence period of 1 to 5 days. The semen characteristics measured were sperm count, semen volume, pre-freeze motility and post-thaw motility. An approach via a decomposition of the variance or via a decomposition of correlation leads essentially to the same conclusions i.e. the within subject factors are not negligible but are less important than the between subject ones. Thus there is broad evidence for overall differences in the quality of the ejaculate from one subject to the other but not for different ejaculates of the same subject.

271 THE EFFECT OF TEMPERATURE ON HUMAN TESTICULAR TISSUE TO OPTIMIZE THE IN VITRO MATURATION OF PRE-FREEZE MOTILITY

The development of intracytoplasmic sperm injection has made the use of testicular sperm a viable option for infertile men with obstructive and nonobstructive azoospermia. Over the past decade, testicular biopsies have been handled and processed using a variety of different methods. Whole biopsy pieces can be effectively cryopreserved in a 10% glycerol diluent (Schiewe et al. 1997 67, S115 abst); however, the ability to find viable, motile sperm post-thaw is improved when prefreeze motility exists. The purpose of this study was to comparatively document in vitro sperm motility enhancement over time at different temperatures, and to prove that an intermediate temperature (28 to 30°C) would optimize sperm longevity for up to 1 week. In this study, 10 men with obstructive azoospermia underwent a surgical, open testicular biopsy procedure. Each biopsy was placed in HEPES buffered-human tubal fluid (mHTF) medium supplemented with 5% human serum albumin (HSA; Irvine Sci., Santa Ana, CA, USA) and transported to the laboratory at room temperature. Each testis biopsy (TBx) was dissected into 8 equal pieces (approximately 2 × 2 × 1 mm). Five intact pieces of TBx were cryopreserved in separate cryovials for future use. The remaining TBx tissue was subdivided into 1 of 3 temperature treatment groups (24, 30, or 37°C) for extended IVM. The 30°C incubation condition was achieved by placing the dish(es) in a Styrofoam box placed on a 37°C warming plate. Each TBx was placed into a separate 100 × 35 mm Falcon dish in 150-μL droplets of mHTF under oil and shredded by needle dissection to disperse the contents of the seminiferous tubules. Reminant tissue was placed into another droplet for additional dissection, as needed. Sperm were analyzed for motility (graded as Type I = twitching, II = undulating, III = slow progression, and IV = rapid progression) at 0, 24, 96, and 144 h without replacing the IVM medium. The percentage increase in motility, compared with 0 h, was statistically contrasted across temperature treatments by chi-squared analysis. Testicular sperm motility ranged from 5 to 25% at 0 h and increased in all groups at 24 h. There was no difference in total motility at 24 h, but progressive motility was higher (P &lt; 0.05) at 37°C compared with 24°C. Significant differences (P &lt; 0.05) were observed in total percentage of motility/percentage of progressive motility at 96 h with treatment differences (*) being 30°C (66%*/44%*) &gt; 24°C (42%*/14%) &gt; 37°C (18%*/10%). This statistical trend continued at 144 h with 30°C (42%*/23%*) &gt; 24°C (24%*/8%) &gt; 37°C (9%*/5%). During this study, a viable pregnancy was achieved using a 30°C sample 8 days post biopsy, exceeding 2 previous healthy triplet pregnancies, which were successful using 4-day-old TBx specimens in 1996 and 2004. This study confirms that an intermediate IVM temperature of 30°C is optimal to enhance TBx cryopreservation, which in our laboratory is generally performed at 24 to 72 h of IVM. Our routine pregnancy success in applying ICSI with IVM, thawed TBx sperm discounts the concern some individuals express regarding DNA fragmentation of sperm over time.

Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen

Six donor semen samples were evaluated after 28 years cryopreservation in liquid nitrogen. The results showed that the samples retained good postthaw motility recovery and normal levels of binding to the human zona pellucida and that four of the five samples tested also gave normal levels of zona-induced acrosome reaction. In conclusion, human sperm can survive very long-term storage, which is pertinent information for clinicians referring boys and young men for sperm banking before chemotherapy.