scholarly journals Improving the Rabbit Semen Cryopreservation Protocol: Comparison Between Two Extenders and Inseminating Doses

2020 ◽  
Vol 20 (3) ◽  
pp. 887-898
Author(s):  
Michele Di Iorio ◽  
Giusy Rusco ◽  
Maria Antonietta Colonna ◽  
Michele Schiavitto ◽  
Maria Silvia D’Andrea ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Artificial Insemination ◽  
Sperm Quality ◽  
Dna Integrity ◽  
Semen Cryopreservation ◽  
Fertilizing Ability ◽  
Cryopreservation Protocol

AbstractThis study has been designed to optimize the semen freezing protocol in rabbits, in this regard we compared a Tris-citrate-glucose (TCG) extender with a commercial one (Cortalap®), that to the best of our knowledge has never been used up to now on the in vitro freezability and fertilizing ability of cryopreserved rabbit semen. Two different inseminating semen doses were considered. Five pooled semen samples were divided into two subsamples and each of them were diluted to a ratio 1:1 (v:v) with a freezing extender composed of TCG or Cortalap® containing 16% of dimethylsulfoxide and 0.1 mol/L of sucrose. The extended semen was filled into 0.25 mL plastic straws and frozen above a liquid nitrogen surface. After thawing (50°C/10 seconds) we determined sperm motility, viability, membrane functionality, acrosome and DNA integrity. Our results showed that the Cortalap® extender significantly improved the in vitro post-thaw sperm quality, in comparison to TCG. When we compared the extenders in vivo, no significant differences in the reproductive performances were observed independently by inseminating doses used. In this study we demonstrated that Cortalap® extender can be used as an alternative to TCG. Thus, the Cortalap® being a ready to use extender, implies a reduction of time, mistakes and microbial contaminations during its preparation. This discovery results as significant because it provides beyond an important contribution to the creation of the first Italian semen cryobank of rabbit breeds and also for livestock rabbit farms based on artificial insemination (AI) program.

scholarly journals Validation of the Turkey Semen Cryopreservation by Evaluating the Effect of Two Diluents and the Inseminating Doses

Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1329
Author(s):  
Michele Di Iorio ◽  
Giusy Rusco ◽  
Roberta Iampietro ◽  
Lucia Maiuro ◽  
Achille Schiavone ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
In Vivo Test ◽  
Sperm Analysis ◽  
Fertilizing Ability ◽  
Vitro Analysis ◽  
Hatching Rates

This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 × 106 sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 × 109 sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis—CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 × 106 sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates.

scholarly journals Optimization of Sperm Cryopreservation Protocol for Mediterranean Brown Trout: A Comparative Study of Non-Permeating Cryoprotectants and Thawing Rates In Vitro and In Vivo

Animals ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 304 ◽  
Author(s):  
Giusy Rusco ◽  
Michele Di Iorio ◽  
Pier Paolo Gibertoni ◽  
Stefano Esposito ◽  
Maurizio Penserini ◽  
...  
Keyword(s):  
Brown Trout ◽  
Semen Quality ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Dna Integrity ◽  
Experimental Conditions ◽  
C 30 ◽  
Thawing Rate

The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

scholarly journals Efficiency of Commercial Egg Yolk-Free and Egg Yolk-Supplemented Tris-Based Extenders for Dromedary Camel Semen Cryopreservation

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 999 ◽  
Author(s):  
Ayman Abdel-Aziz Swelum ◽  
Islam M. Saadeldin ◽  
Hani Ba-Awadh ◽  
Mohsen G. Al-Mutary ◽  
Abdullah F. Moumen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Dromedary Camel ◽  
Plasma Membrane Integrity ◽  
Semen Cryopreservation

This study compared the efficiency of commercial egg yolk-free (AndroMed, OPTIXcell) and egg yolk-supplemented (Triladyl, Steridyl) Tris-based extenders for semen cryopreservation in seven adult dromedary camels. The camel-specific extender SHOTOR was used as control. The collected semen samples were evaluated and diluted with SHOTOR, Triladyl, Steridyl, AndroMed, or OPTIXcell. The diluted semen was gradually cooled and equilibrated for two hours before liquid nitrogen freezing. Semen was evaluated prior to freezing and after freeze-thawing cycles for motility, kinetics, vitality, abnormality, plasma membrane integrity, and DNA fragmentation using computer-assisted sperm analysis. In pre-freezing evaluation, progressive sperm motility was higher in SHOTOR-diluted samples (21.54 ± 1.83) than in samples diluted with Steridyl, OPTIXcell, or AndroMed (15.76 ± 1.80, 17.43 ± 1.10, and 13.27 ± 1.07, respectively). Moreover, Triladyl and SHOTOR resulted in significantly (p < 0.05) better sperm vitality and DNA integrity than all other diluents, but Triladyl resulted in a significantly (p < 0.05) better plasma membrane integrity (87.77 ± 0.31) than SHOTOR (85.48 ± 0.58). In the post-thawing evaluation, Triladyl led to significantly (p < 0.05) higher sperm motility (38.63 ± 0.81%; p < 0.05) when compared to SHOTOR, Steridyl or AndroMed (35.09 ± 1.341%, 34.4 ± 0.84%, and 31.99 ± 1.48%, respectively), with OPTIXcell being the least efficient (28.39 ± 0.86%). Progressive sperm motility was the highest when using Triladyl. Post-thawing curvilinear, straight line and average path sperm velocities were highest with Triladyl and lowest with AndroMed. Triladyl led to the highest linearity coefficient and straightness sperm coefficient, while SHOTOR to the highest DNA and plasma membrane integrity. OPTIXcell and AndroMed resulted in poor post-thawing sperm vitality, while Steridyl was less efficient than Triladyl. The highest rate of sperm abnormalities was recorded with OPTIXcell and the lowest with SHOTOR or Triladyl. In conclusion, SHOTOR, Triladyl, Steridyl, AndroMed, and OPTIXcell can all be used for camel semen cryopreservation; however, SHOTOR and Triladyl provided the best post-thawing sperm quality. Based on our findings, Triladyl is the best commercially available extender for dromedary camel semen cryopreservation to date.

scholarly journals A Simple and Efficient Semen Cryopreservation Method to Increase the Genetic Variability of Endangered Mediterranean Brown Trout Inhabiting Molise Rivers

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 403
Author(s):  
Giusy Rusco ◽  
Michele Di Iorio ◽  
Roberta Iampietro ◽  
Stefano Esposito ◽  
Pier Paolo Gibertoni ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Brown Trout ◽  
Sperm Concentration ◽  
Mediterranean Area ◽  
Fertilization Rate ◽  
Semen Cryopreservation ◽  
Nitrogen Vapor ◽  
C 30

The aim of our study was to test the effectiveness of a simple semen cryopreservation procedure, developed for cultivated salmonid, on the wild salmonid of the Mediterranean area and to evaluate the effect of different thawing rates and sperm-to-egg ratios. The semen of five individual males was diluted into a final extender concentration of 0.15 M glucose and 7.5% methanol and loaded into 0.25 mL plastic straws, and a final sperm concentration of 3.0 × 109 sperm/mL was obtained. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above the liquid nitrogen level for 5 min. The semen was thawed at 40 °C/5 s or 10 °C/30 s. The sperm cryosurvival was evaluated by examining in vitro the sperm motility parameters using the CASA system, followed by fertilization trials in vivo, using three different sperm-to-egg ratios 6 × 105, 4.5 × 105 and 3 × 105:1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 °C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 × 105:1. According to these outcomes, the cryopreservation procedure that was tested turned out to be effective for the wild population of Mediterranean brown trout and practical for the creation of the first European semen cryobank foreseen as part of our “LIFE” Nat.Sal.Mo. project.

scholarly journals The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3452
Author(s):  
Uchechi Linda Ohaneje ◽  
Uchebuchi Ike Osuagwuh ◽  
Manuel Alvarez-Rodríguez ◽  
Iván Yánez-Ortiz ◽  
Abigail Tabarez ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Mitochondrial Activity ◽  
Dna Integrity ◽  
Vitro Fertilization

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

Effect of a pre-freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics

2013 ◽  
Vol 25 (6) ◽  
pp. 935 ◽  
Author(s):  
C. Tomás ◽  
E. Blanch ◽  
A. Fazeli ◽  
E. Mocé
Keyword(s):  
Dna Fragmentation ◽  
Sperm Quality ◽  
Epithelial Cell Line ◽  
Dna Integrity ◽  
Sperm Longevity ◽  
Boar Sperm ◽  
Fragmentation Dynamics ◽  
Freezing Treatment

The aim of this work was to examine how a pre-freezing treatment with cholesterol-loaded cyclodextrins (CLC) affects boar sperm longevity, capacitation dynamics, ability to bind to a porcine telomerase-immortalised oviductal epithelial cell line (TERT-OPEC) in vitro and DNA integrity dynamics after freeze–thawing. Although the samples treated with CLC exhibited lower sperm quality than the control samples (P < 0.05) immediately after thawing, these differences disappeared (P > 0.05) after long-term incubation (26 h at 37 or 16°C). Additionally, the CLC-treated spermatozoa underwent similar capacitation and DNA fragmentation dynamics as the control spermatozoa (P > 0.05). However, CLC-treated spermatozoa were better able to bind to TERT-OPEC in vitro (P < 0.0001). In conclusion, the pre-freezing treatment of boar spermatozoa with CLC enhanced the ability of the spermatozoa to bind to TERT-OPEC in vitro, which could have an effect on the establishment of the sperm reservoir in the ampullary­–isthmic junction in vivo. Additionally, frozen–thawed spermatozoa can be stored at 16°C for at least 6 h without a significant observable decline in sperm quality, which could be beneficial for the transport of thawed diluted doses of spermatozoa from the laboratory to the farm.

scholarly journals The effects of xanthan gum on equine sperm quality during cooling storage

2019 ◽  
Vol 71 (1) ◽  
pp. 28-34
Author(s):  
S.M.M. Gheller ◽  
C.D. Corcini ◽  
F.C.C. Santos ◽  
G.C. Tavares ◽  
V.G.G. Costa ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Xanthan Gum ◽  
Sperm Quality ◽  
Control Group ◽  
Dna Integrity ◽  
Control Groups ◽  
Cooling Period ◽  
Semen Extender ◽  
And Control

ABSTRACT This study was designed to evaluate the possible benefits of adding xanthan gum to a standard extender for equine through in vitro analyzes of sperm quality. Semen was collected four times from five different stallions (n= 20 samples) and subjected to cooled storage under different conditions: control (only standard extender) and three different concentrations of xanthan gum (0.01%, 0.12%, and 0.25%) supplemented to the extenders. Sperm parameters, such as motility, mitochondrial functionality, and membrane, acrosome, and DNA integrity were measured after 0h, 24h, 48h, and 72h of sperm storage at 5ºC. Our observations indicated that sperm motility declined with longer cooling period with the 0.25% xanthan gum supplementation group compared with the control group. Other parameters, such as mitochondrial functionality and membrane and acrosome integrity also declined for all treatments during storage; however, no differences were observed between xanthan gum and control groups. DNA integrity did not significantly change during the storage. In conclusion, the addition of xanthan gum to equine semen extender is not harmful to the sperm structure, despite reducing the sperm motility.

An Insight into Novel Sperm Cell Proteins as Bio-markers for Male Infertility: A Review

2021 ◽  
Vol 21 ◽  
Author(s):  
Naina Kumar ◽  
Namit Kant Singh
Keyword(s):  
Male Infertility ◽  
Sperm Motility ◽  
Zona Pellucida ◽  
English Language ◽  
Semen Analysis ◽  
Meta Analysis ◽  
Sperm Dna Damage ◽  
Sperm Proteins

: Male infertility is rising now-a-days and accounts for major part of infertility cases worldwide. Novel tests are being developed for better detection and management of male infertility. Though there are many tests available for diagnosing male infertility like acrosome reaction rate, hemizona assay, in vivo or in vitro sperm penetration assay, sperm DNA damage tests, but semen analysis is most commonly used initial test for male infertility. It is usually associated with failure to detect cause in many cases, as seminal composition gets affected by a number of factors and can give false reports. Furthermore, it does not give any information about defects in capacitation, sperm Zona Pellucida interaction and sperm’s ability to fertilize oocytes. This results in failure of detection and delayed management of male infertility. Hence, the present review was conducted to identify various sperm proteins that play significant role in spermatogenesis, sperm motility, sperm-Zona Pellucida interaction and fertilization. These proteins can be used in future as markers of male infertility and will aid in better detection and management of male infertility. Methodology: Search for literature was made from 1970 to 2020 from various databases like PUBMED, SCOPUS, Google Scholar on sperm proteins and their role in male fertility using keywords: “sperm protein as bio-markers”, “novel sperm proteins as markers of infertility”, “Sperm proteins essential for capacitation, sperm motility and oocyte fertilization”. Inclusion criteria: All full-length research articles, systematic reviews, meta-analysis or abstracts on sperm proteins and male infertility published in English language in peer-reviewed journals were considered.

scholarly journals Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Reproduction ◽  
2004 ◽  
Vol 127 (5) ◽  
pp. 547-556 ◽  
Author(s):  
R E Spindler ◽  
Y Huang ◽  
J G Howard ◽  
P Wang ◽  
H Zhang ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Giant Panda ◽  
Calcium Ionophore ◽  
Sperm Cryopreservation ◽  
Management Tools ◽  
Giant Pandas ◽  
Before And After ◽  
Cryopreservation Protocol ◽  
Acrosomal Integrity

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze–thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (− 40 and − 100 °C/min) cryopreservation by incubation in HEPES-buffered Ham’s F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean±s.e.m. sperm motility post-thaw (56.1 ± 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 ± 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 ± 1.7% versus cryopreserved–thawed, 81.7 ± 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 ± 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 ± 1.1%). Frozen–thawed sperm preincubated without accelerators underwent capacitation (49.6 ± 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 ± 1.4%) and without accelerators (9 h: 41.2 ± 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.

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