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<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<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.

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Bivalent response to long-term storage in liquid-preserved boar semen: A flow cytometric analysis

Cytometry Part A ◽

10.1002/cyto.a.22058 ◽

2012 ◽

Vol 81A (7) ◽

pp. 576-587 ◽

Cited By ~ 20

Author(s):

Heiko Henning ◽

Anna M. Petrunkina ◽

Robin A. P. Harrison ◽

Dagmar Waberski

Keyword(s):

Flow Cytometric Analysis ◽

Long Term Storage ◽

Flow Cytometric ◽

Boar Semen ◽

Term Storage

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Cryopreservation of boar semen

Czech Journal of Animal Science ◽

10.17221/47/2020-cjas ◽

2020 ◽

Vol 65 (No. 4) ◽

pp. 115-123

Author(s):

Marija Jovičić ◽

Eva Chmelíková ◽

Markéta Sedmíková

Keyword(s):

Animal Species ◽

Semen Quality ◽

Egg Yolk ◽

High Rate ◽

Freezing And Thawing ◽

Long Term Storage ◽

Boar Semen ◽

Boar Spermatozoa ◽

Term Storage

Sperm cryopreservation is the best technology for long-term storage of the semen. However, the damage of boar spermatozoa by cryopreservation is more severe than in other animal species and a standardized freezing protocol for efficient cryopreservation has not been established yet. Semen quality and freezability vary greatly between breeds as well as between individual boars and even the season. Boar spermatozoa are sensitive to low temperatures; they sustain damage and a high rate of mortality and freezing/thawing the boar semen may strongly impair the sperm function and decrease the semen quality. The freezability of boar semen can be influenced by a cryopreservation procedure, and also by using various additives to freezing and thawing extenders such as antioxidants. In order to obtain acceptable results after thawing the boar semen, it is necessary to combine an optimal amount of additives (glycerol, egg yolk, sugars, antioxidants), cooling and warming velocities.

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Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus)

Reproduction Fertility and Development ◽

10.1071/rd05108 ◽

2006 ◽

Vol 18 (3) ◽

pp. 319 ◽

Cited By ~ 63

Author(s):

J. K. O'Brien ◽

T. R. Robeck

Keyword(s):

Tursiops Truncatus ◽

Sperm Quality ◽

Bottlenose Dolphins ◽

Sperm Concentration ◽

Motility Index ◽

Liquid Storage ◽

Directional Freezing

Research was conducted to develop sperm sorting and novel sperm preservation methodologies for sex predetermination in the bottlenose dolphin (Tursiops truncatus) using artificial insemination. In Study 1, the effect of seminal plasma (SP), sperm concentration and freezing rate (FR) on in vitro sperm quality of liquid-stored, non-sorted spermatozoa was examined. There was no effect (P > 0.05) of prefreeze SP addition on post-thaw quality (progressive motility, kinetic rating, sperm motility index (SMI), viability and acrosome integrity). Post-thaw motility parameters and viability were higher (P < 0.05) for slow FR than fast FR samples. In Study 2 investigating the effects of liquid storage and sorting on sperm quality, motility and SMI after sorting and centrifugation were lower (P < 0.05) than those of the initial ejaculate. The sort rate for enrichment (91 ± 4% purity) of X- and Y-bearing spermatozoa was 3400 ± 850 spermatozoa sex−1 s−1. In Study 3, compared with a modified straw method, directional freezing resulted in enhanced in vitro quality of sorted and non-sorted spermatozoa derived from liquid-stored semen (P < 0.05). In Study 4, endoscopic insemination of three dolphins with sorted, frozen–thawed X-bearing spermatozoa resulted in one conception and the birth of a female calf. High-purity sorting of dolphin spermatozoa, derived from liquid-stored semen, can be achieved with minimal loss of in vitro sperm quality and samples are functional in vivo.

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Temperature Effects on Interspecific Hybridization between Gladiolus ×grandiflora and G. tristis

HortScience ◽

10.21273/hortsci.36.2.341 ◽

2001 ◽

Vol 36 (2) ◽

pp. 341-343 ◽

Cited By ~ 4

Author(s):

Yasumasa Takatsu ◽

Masakazu Kasumi ◽

Toru Manabe ◽

Mikio Hayashi ◽

Eiichi Inoue ◽

...

Keyword(s):

Interspecific Hybridization ◽

Wild Species ◽

Storage Temperature ◽

Pollen Viability ◽

Flow Cytometric Analysis ◽

Morphological Data ◽

Modern Cultivar ◽

Long Term Storage ◽

Flow Cytometric

Interspecific hybridization between a modern cultivar of Gladiolu×grandiflora hort. (2n = 60) and the wild species G. tristis L. (2n = 30) was made to introduce characteristics of the wild species into the cultivated one. Gladiolus ×grandiflora is a summer-flowering species, and G. tristis flowers in winter. The effect of storage temperature on pollen viability was tested, as long-term storage of pollen was necessary to facilitate crossing these two species. Pollen of G. tristis could be stored at -20 °C for ≈1 year, and was more practical than storage at -80 °C. Air temperature affected pollen tube growth, fertility, and fruit set in the cross between G. ×grandiflora and G. tristis, and low temperatures (15 to 20 °C) were best. The morphological data and flow cytometric analysis showed that the F1 plants were hybrids between G. ×grandiflora and G. tristis.

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201 EFFECTS OF REMOVING SEMINAL PLASMA DURING 8-h STORAGE BEFORE SEX-SORTING BOVINE SPERM

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab201 ◽

2012 ◽

Vol 24 (1) ◽

pp. 213 ◽

Cited By ~ 1

Author(s):

C. A. Burroughs ◽

K. M. Evans ◽

R. W. Lenz ◽

G. E. Seidel

Keyword(s):

Seminal Plasma ◽

Sperm Concentration ◽

Computer Assisted ◽

Sperm Analysis ◽

Long Term Storage ◽

Bovine Sperm ◽

Standard Procedures ◽

Term Storage ◽

Artificial Vagina

We evaluated sex-sorting parameters and post-thaw motility for sperm stored with or without seminal plasma for 8 h before sorting. One first ejaculate was collected from each of 6 bulls routinely collected via artificial vagina; ejaculates contained at least 70% motile and 75% morphologically normal sperm and sperm concentrations ranged from 0.75 to 2.21 × 109 sperm mL–1. Ejaculates were divided into 2 samples and centrifuged at 1000 × g for 15 min. Seminal plasma from 1 sample was replaced with TALP (pH 7.4) to a sperm concentration of 1.4 × 109 sperm mL–1. The seminal plasma/sperm admixture of the other sample (control) was suspended to initial ejaculate sperm concentration. Both samples were stored for 8 h at 16°C before being subjected to standard sex-sorting procedures. Sperm were analyzed and bulk sorted on a MoFlo SX (XY Inc., Navasota, TX, USA) flow cytometer/cell sorter for percentage of live-oriented cells, percentage of membrane-impaired sperm (cell membranes permeable to red food colouring, which were discarded during sorting) and resolution between X- and Y-bearing sperm populations (peak to valley ratio). Sorted sperm were frozen according to standard procedures and post-thaw motility was determined immediately after thawing using computer-assisted sperm analysis. Treatments were compared using a paired t-test. Control sperm stored with seminal plasma resulted in a higher percentage of live-oriented cells (55%) versus those stored without seminal plasma (51%; P = 0.02). The percentage of membrane-impaired sperm was lower for control sperm (19%) than that of samples without seminal plasma (28%; P < 0.001). Resolution was greater for sperm stored without seminal plasma (34%) than for control sperm (10%; P = 0.04). Post-thaw, both total and progressively motile sperm were higher for samples without seminal plasma (63 and 53%, respectively) compared with those of the control samples (52 and 45%, respectively; P < 0.04). In conclusion, sperm stored for 8 h without seminal plasma had greater resolution between X- and Y-bearing populations and higher post-thaw motility than control sperm. However, these samples had a higher percentage of membrane-impaired sperm that were removed during sorting. Long-term storage of sperm in their seminal plasma before sex-sorting appears to be detrimental to post-sorting, post-thaw sperm motility.

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89. Application of cryopreservation techniques for long-term storage of clinical isolates of Candida fungi

Cryobiology ◽

10.1016/j.cryobiol.2010.10.093 ◽

2010 ◽

Vol 61 (3) ◽

pp. 388-389

Author(s):

A. Yu Artuyants ◽

V.F. Martsenyuk ◽

I.P. Vysekantsev

Keyword(s):

Clinical Isolates ◽

Long Term Storage ◽

Cryopreservation Techniques ◽

Term Storage

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Long term storage of Pleurotus ostreatus and Trametes versicolor isolates using different cryopreservation techniques and its impact on laccase activity

Fungal Biology ◽

10.1016/j.funbio.2015.10.004 ◽

2015 ◽

Vol 119 (12) ◽

pp. 1345-1353 ◽

Cited By ~ 2

Author(s):

Ivana Eichlerová ◽

Ladislav Homolka ◽

Michal Tomšovský ◽

Ludmila Lisá

Keyword(s):

Pleurotus Ostreatus ◽

Trametes Versicolor ◽

Laccase Activity ◽

Long Term Storage ◽

Cryopreservation Techniques ◽

Term Storage

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THE USE OF DORMANT-BUD CRYOPRESERVATION FOR LONG-TERM STORAGE OF PECAN AND WALNUT GERMPLASM

HortScience ◽

10.21273/hortsci.25.9.1142c ◽

1990 ◽

Vol 25 (9) ◽

pp. 1142c-1142 ◽

Cited By ~ 1

Author(s):

Todd M. Morrissey ◽

William A. Gustafson

Keyword(s):

Woody Plants ◽

Vaccinium Corymbosum ◽

Black Walnut ◽

Long Term Storage ◽

Dormant Bud ◽

If Current ◽

Cryopreservation Techniques ◽

Long Term Preservation ◽

Term Storage

A study was designed to determine if current dormant-bud cryopreservation techniques investigated on woody plants, such as apple (Malus domestica), gooseberry (Ribes), blueberry (Vaccinium corymbosum) and pear (Pryus communis) etc., could be applied to certain nut tree species for long-term preservation. Pecan (Carya illinoinensis) and black walnut (Juglans nigra) were exposed to prefreezing temperatures ranging from -10° C to -40° C and then directly immersed in liquid nitrogen for 2 hrs. Dehydration by prefreezing was not sufficient for bud survival in pecan. Bud survival was increased by dehydrating stem sections prior to prefreezing. Prefreezing at -30° or -40° C was suitable for survival of black walnut.

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59 THE INFLUENCE OF DILUENT EGG YOLK PARTICLES ON THE ACCURACY OF FLOW CYTOMETRIC ANALYSES OF SPERM FUNCTIONAL CHARACTERISTICS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab59 ◽

2009 ◽

Vol 21 (1) ◽

pp. 130

Author(s):

A. K. Alhaider ◽

P. F. Watson

Keyword(s):

Plasma Membrane ◽

Membrane Fluidity ◽

Flow Cytometric Analysis ◽

Egg Yolk ◽

High Plasma ◽

Plasma Membrane Fluidity ◽

True Value ◽

Flow Cytometric ◽

Percentage Points ◽

Method Agreement

Flow cytometry is an important tool to assess semen quality and sperm function, especially after cryopreservation, but the presence of egg yolk particles in many semen diluents can distort the results. The aims of the present study were to investigate the effect of the presence of egg yolk in the semen diluent on the outcome of flow cytometric analysis of dog spermatozoa and to explore the use of Laser Dye Styryl-751 (LDS-751), a permeant DNA stain with an emission wavelength of 712 nm, to exclude egg yolk particles from the analysis of plasma membrane fluidity (Merocyanine 540 and Yo-Pro-1) and intracellular Ca2+ (Fluo-3 and PI). Flow cytometric analyses were carried out on a Coulter Epics XL flow cytometer (Beckman-Coulter Inc., Fullerton, CA). Data were analyzed using EXPO32TM ADC software (Beckman-Coulter). A paired t-test was used to test whether gating out egg yolk particles using LDS-751 affected the analysis. Method agreement analysis was undertaken to assess the agreement between the 2 staining protocols (i.e. with or without LDS-751). The proportion of spermatozoa with high plasma membrane fluidity was significantly (P < 0.05) greater after gating out egg yolk particles; the measurements of high membrane fluidity spermatozoa were on average 7.7 percentage points greater than without gating out egg yolk as shown by the method agreement analysis (SD = 9.35). Similarly, the proportion of spermatozoa with high intercellular Ca2+ was significantly (P < 0.01) greater after gating out egg yolk particles; the proportions of spermatozoa with high intracellular Ca2+ were 5.95 percentage points greater when egg yolk particles were gated out compared with measurements in the presence of egg yolk particles (SD = 5.43). The method agreement shows that both methods show close agreement with and without egg yolk particles (i.e. results mostly fall within the mean of differences between pairs of repeated measurements ±2 SD). Therefore, the 2 methods are measuring the same phenomena. However, the change in measurement values with exclusion of egg yolk particles is significant. Therefore, accuracy is increased; that is, closer to the true value. In conclusion, using MC540/Yo-Pro-1 and Fluo-3/PI staining alone in the presence of egg yolk particles resulted in underestimation of the proportions of high plasma membrane fluidity and high intracellular Ca2+ spermatozoa, respectively. LDS-751 could be used with other fluorescent stains that target organelles such as mitochondria and acrosomes with multicolor flow cytometric analysis to exclude egg yolk particles.

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