Preservation of beluga (Delphinapterus leucas) spermatozoa using a trehalose-based cryodiluent and directional freezing technology

2010 ◽  
Vol 22 (4) ◽  
pp. 653 ◽  
Author(s):  
J. K. O'Brien ◽  
T. R. Robeck
Keyword(s):  
Freezing Rate ◽  
Slow Freezing ◽  
Delphinapterus Leucas ◽  
Freezing Method ◽  
Progressive Motility ◽  
Sperm Characteristics ◽  
Preservation Method ◽  
Systematic Development ◽  
Directional Freezing

A beluga (Delphinapterus leucas) sperm preservation method was developed for use in genome banking and AI. In Study 1, glycerol-based cryodiluents (modified BF5F and modified Platz Diluent Variant (PDV)) were unable to maintain adequate progressive motility using straws (fast and slow freezing rate (FR)) or pellets (slow FR). Neither freezing method nor FR affected in vitro sperm characteristics (P > 0.05), but retention of prefreeze progressive motility following thawing was greater (P < 0.05) for BF5F (21%) than PDV (15%). In Study 2, examining the effects of straw freeze–thawing using BF5F with glycerol (1 and 3%, v/v) or trehalose (46 and 91 mM) on sperm characteristics, samples cryopreserved in trehalose exhibited superior (P < 0.05) in vitro parameters compared with their glycerol-treated counterparts. In Study 3, compared with a straw method, directional freezing using 91 mM trehalose enhanced (P < 0.05) sperm characteristics, with samples retaining 38%, 75% and 61% of their prefreeze progressive motility, curvilinear velocity and viability, respectively. A higher (P < 0.05) proportion of motile spermatozoa displayed rapid velocity after directional (21 ± 1%) compared with straw (12 ± 3%) freezing. Systematic development of a cryodiluent and the use of directional freezing resulted in beluga spermatozoa exhibiting adequate post-thaw quality for genome banking and use in AI.

Semen collection, characterisation and artificial insemination in the beluga (Delphinapterus leucas) using liquid-stored spermatozoa

2008 ◽  
Vol 20 (7) ◽  
pp. 770 ◽  
Author(s):  
J. K. O'Brien ◽  
K. J. Steinman ◽  
T. Schmitt ◽  
T. R. Robeck
Keyword(s):  
Artificial Insemination ◽  
Storage Temperature ◽  
Sperm Concentration ◽  
Serum Testosterone ◽  
Delphinapterus Leucas ◽  
Short Term ◽  
Preservation Method ◽  
First Time ◽  
Pregnancy And Birth

Ejaculates were collected from a beluga (Delphinapterus leucas) to gain an understanding of sperm biology and develop a short-term sperm preservation method for use in artificial insemination (AI). Ejaculate parameters and biochemistry, semen production and serum testosterone concentrations of an adult male were characterised for 21 months. Sperm viability, acrosome integrity and morphology did not change (P > 0.05) but ejaculate volume, sperm concentration and total spermatozoa per ejaculate were higher (P < 0.05) from January to June than from July to December. Peak testosterone concentrations (P < 0.05) were observed from October to April (8.0 ± 1.6 ng mL–1). The effects of hyaluronic acid (HA), antioxidants, storage temperature and time on in vitro sperm characteristics were examined. Motility parameters and viability were improved (P < 0.05) when semen was stored at 5°C compared with 21°C. During the first 24 h of storage sperm agglutination was absent only at 5°C in the presence of HA. A nulliparous 28-year-old female was inseminated endoscopically with liquid-stored semen. A pregnancy and birth of a calf was achieved following AI for the first time in this species, thereby validating both the AI technique and the fertility of beluga spermatozoa after chilled storage in a specialised diluent.

Characteristics of high-quality Asian elephant (Elephas maximus) ejaculates and in vitro sperm quality after prolonged chilled storage and directional freezing

2013 ◽  
Vol 25 (5) ◽  
pp. 790 ◽  
Author(s):  
J. K. O'Brien ◽  
K. J. Steinman ◽  
G. A. Montano ◽  
C. C. Love ◽  
R. L. Saiers ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Elephas Maximus ◽  
Mitochondrial Activity ◽  
Dna Integrity ◽  
High Quality ◽  
Chilled Storage ◽  
Progressive Motility ◽  
Directional Freezing

The in vitro quality of spermatozoa from one elephant (Elephas maximus) was examined after chilled storage and directional freezing (DF). High-quality, non-contaminated ejaculates (77.6 ± 6.0% progressive motility, 3.9 ± 1.5 µg creatinine mL–1 raw semen, 2.7 ± 0.6% detached heads) were cryopreserved after 0 (0hStor), 12 (12hStor) and 24 h (24hStor) of chilled storage. At 0 h and 6 h post-thawing, total motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and normal morphology were similar (P > 0.05) across treatments. In contrast, progressive motility, rapid velocity and several kinematic parameters were lower (P < 0.05) for 24Stor compared with 0hStor at 0 h post-thaw. By 6 h post-thaw, amplitude of lateral head displacement and velocity parameters (average pathway, straight-line and curvilinear velocity) were lower (P < 0.05) for 24hStor compared with 0hStor and 12hStor. DNA integrity was high and remained unchanged (P > 0.05) across all groups and processing stages (1.6 ± 0.6% of cells contained fragmented DNA). Results indicate that DF after up to 12 h of chilled storage results in a post-thaw sperm population of acceptable quality for artificial insemination. These findings have implications for the cryopreservation of sex-sorted spermatozoa, which typically undergo more than 12 h of chilled storage prior to sorting and preservation.

118 CRYOPRESERVATION OF CONVENTIONAL AND SEX-SORTED BULL SPERM USING A DIRECTIONAL FREEZING METHOD

2007 ◽  
Vol 19 (1) ◽  
pp. 176 ◽  
Author(s):  
H. Hayakawa ◽  
T. Yamazaki ◽  
M. Oshi ◽  
M. Hoshino ◽  
O. Dochi ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Interface Velocity ◽  
Egg Yolk ◽  
Sperm Viability ◽  
Freezing Method ◽  
Progressive Motility ◽  
Nitrogen Vapor ◽  
Bull Sperm ◽  
Directional Freezing ◽  
Acrosomal Integrity

The objective of this study was to find an optimal parameter (liquid-ice interface velocity; velocity) of the directional freezing method (Arav et al. 2002 Reprod. Nutr. Dev. 42, 583–586) for conventionally processed bull sperm stored in 0.5-mL straws. We also evaluated sex-sorted bull sperm frozen using this method. Experiment 1: Each single ejaculate from two Holstein bulls was split and processed in egg yolk citrate extender (2 steps, 6% glycerol final; EYC) or egg yolk Tris extender (1 step, 7% glycerol final; EYT1). After cooling and loading in 0.5-mL plastic straws, each batch of semen was frozen using a directional 0.5-mL straw freezer (MTG 550; IMT, Ltd., Ness-Ziona, Israel) in 6 different velocities (v2.0, v2.2, v2.4, v2.6, v2.8, v3.0; mm s-1). Ice seeding time was 45 s. Static freezing in liquid nitrogen vapor was used as the control. Sperm parameters were measured using computer-assisted semen analysis (CASA) at 0, 1, and 2 h after thawing. The thawed samples were also evaluated for sperm viability and acrosomal integrity by triple staining. The experiment was repeated 3 times. Data were analyzed by ANOVA. No significant differences in general and progressive motility were observed in each extender regardless of the freezing method. However, sperm viability and acrosomal integrity of sperm frozen in EYC were highest at v3.0 (73.2% and 84.7%, respectively) and were generally higher after MTG freezing (63.4 to 73.2% and 80.6 to 84.7%, respectively) than in the control (52.6% and 73.1%, respectively; P &lt; 0.05 or 0.01) except for v2.4 (54.5% and 70.8%). Sperm viability (74.6 to 77.9% vs. 63.2%) and acrosomal integrity (84.8 to 88.9% vs. 79.3%) in EYT after MTG freezing were also higher than in the control (P &lt; 0.01). Experiment 2: Two single ejaculates from a Holstein bull were flow cytometrically sex-sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) for X sperm. Each ejaculate was assigned to processing in EYC or egg yolk Tris extender (2 steps, 6% glycerol final; EYT2). Processed sperm was frozen using MTG 550 (velocity set at v3.0) or in liquid nitrogen vapor as control and analyzed as in Experiment 1. In the control, general (40.2% vs. 21.1%; P &lt; 0.01) and progressive (16.3% vs. 4.3%; P &lt; 0.05) motility in EYC were higher than in EYT2, respectively, at 0 h. In MTG freezing, motility (at 0 h, 42.0% vs. 28.3%; P &lt; 0.05), viability (60.5% vs. 40.8%; P &lt; 0.01), and acrosomal integrity (86.9% vs. 71.0%; P &lt; 0.05) in EYC were higher than in EYT2, respectively. But in experiment 2, there were no differences in motility, progressive motility, viability, and acrosomal integrity between MTG freezing and control. The above results suggest that MTG freezing improves membrane and acrosomal quality of bull sperm frozen in 0.5-mL straws. Faster interface velocity (3.0 mm s-1) seems optimal for the given condition. Egg yolk citrate extender seems to be beneficial for MTG freezing of sex-sorted bull sperm, but further studies using ejaculates from different bulls are required to confirm the apparent benefit.

scholarly journals Cryopreservation of Bălțată cu Negru Românească Cattle Immature Oocytes by Slow Freezing Method: Preliminary Research

2016 ◽  
Vol 49 (4) ◽  
pp. 107-113
Author(s):  
S.I. Borş ◽  
Șt. Creangă ◽  
D.L. Dascălu ◽  
Mirela Ariton ◽  
Andra-Sabina Neculai-Văleanu ◽  
...  
Keyword(s):  
Transvaginal Ultrasound ◽  
Spherical Shape ◽  
Cumulus Cells ◽  
Slow Freezing ◽  
Freezing Method ◽  
Multiple Ovulation ◽  
Preliminary Research ◽  
Follicular Puncture ◽  
Bovine Oocytes

Abstract Research on bovine oocytes cryopreservation is important for successful preservation of genetically valuable animal. The transvaginal ultrasound-guided follicular puncture coupled with in vitro embryo production has become competitive and alternative method for MOET (Multiple ovulation and embryo transfer) in dairy cattle. The aim of this preliminary research is to presents the result of Bălțată cu Negru Românească (BNR) cows oocytes recovery by two different protocols and its cryopreservation by slow freezing method. By applying the recovery oocytes from slaughterhouse ovary we obtained an average of 16.34 ± 6.71 oocytes per cow, much higher compared with the Ovum Pick Up (OPU) method, which reveals an average of 2.75 ± 0.2 oocytes per cow. After applying the slow freezing procedures using the ethylene glycol cryoprotectant we observed the oocytes with cumulus cells normal with the spherical shape and normal zone pellucida.

31 Evaluation of in vitro-produced bovine embryos with conventional and SexedULTRA-4M X and Y chromosome-bearing semen: survival after slow freezing for direct transfer

2022 ◽  
Vol 34 (2) ◽  
pp. 250
Author(s):  
H. Álvarez-Gallardo ◽  
M. Kjelland ◽  
M. Pérez-Martínez ◽  
A. Velázquez-Roque ◽  
F. Villaseñor-González ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Slow Freezing ◽  
Direct Transfer ◽  
Bovine Embryos

Directional freezing of spermatozoa and embryos

2014 ◽  
Vol 26 (1) ◽  
pp. 83 ◽  
Author(s):  
Amir Arav ◽  
Joseph Saragusty
Keyword(s):  
Cooling Rate ◽  
Drop Size ◽  
Mechanical Damage ◽  
Rapid Freezing ◽  
Wild Animals ◽  
Microscope Slide ◽  
Freezing Method ◽  
Wide Range ◽  
Sexed Semen ◽  
Directional Freezing

Directional freezing is based on a simple thermodynamic principle whereby the sample is moved through a predetermined temperature gradient at a velocity that determines the cooling rate. Directional freezing permits a precise and uniform cooling rate in small- and large-volume samples. It avoids supercooling and reduces mechanical damage caused by crystallisation. Directional solidification was used to date for slow and rapid freezing, as well as for vitrification of oocytes and embryos by means of the minimum drop size technique: small drops are placed on a microscope slide that is moved at high velocity from the hot base to the cold base. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The bovine sexed semen industry may benefit from the increased survival of spermatozoa after directional freezing.

scholarly journals Effect of In-Vitro Alpha Lipoic Acid Addition on Spermatozoa Motility in Sperm Preparation Process

2021 ◽  
Vol 55 (4) ◽  
pp. 246
Author(s):  
Gede Wira Buanayuda ◽  
Hamdani Lunardhi ◽  
Indra Gusti Mansur
Keyword(s):  
Lipoic Acid ◽  
Intrauterine Insemination ◽  
Control Group ◽  
Infertile Couple ◽  
Preparation Process ◽  
Alpha Lipoic Acid ◽  
Sperm Preparation ◽  
Progressive Motility ◽  
Child Hospital

Infertility is a problem for husband and wife, in the last 20 years the number of infertile couples has tended to increase by around 6.5 million pairs. The infertile couple can use the intrauterine insemination method to obtain offspring if a conventional method approach cannot be performed. Insemination requires a sperm preparation stage in which there are centrifugation and resuspension procedures that tend to produce excess reactive oxygen species (ROS). Excessive ROS will damage the motility of the spermatozoa. This study aims to prove the addition of alpha lipoic acid (ALA) as an antioxidant in the process of sperm preparation to improve and maintain better sperm motility. This research is a laboratory study with an experimental research design. The sample consisted of 10 infertile men who visited the Andrology section of the Sayyidah Jakarta Mother and Child Hospital (RSIA), where each ejaculate from the patient would be divided into 3 groups namely (k1) fresh semen as a control group, (k2) sperm preparation group without ALA, (k3) group of sperm preparation with the addition of ALA. The motility of spermatozoa was observed with the WHO 1999 method for 4 hours in units of percent. Progressive motility in k3 (47.95 ± 3.617) was higher than in k2 (38.05 ± 3.278) statistically significantly different after 3 hours of observation (p<0.0001). Progressive motility in k3 (78.8 ± 5.841) was higher than k1 (56.55 ± 7.511) from the initial observation (p <0.0001). The progressive motility of k2 (76.05 ± 6.768) was higher than k1 (56.55 ± 7.511) from the start of the observation (0.0001). It can be concluded that the addition of ALA in the sperm preparation process increases and maintains progressive motility that is better than sperm preparation without ALA addition after 3 hours of observation.

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