scholarly journals Vital Analysis of Cryopreserved Sperm of Marbled Flounder, Pseudopleuronectes yokohamae

2021 ◽  
Vol 12 ◽  
Author(s):  
Shaharior Hossen ◽  
Soo Cheol Kim ◽  
Yusin Cho ◽  
Kang Hee Kho
Keyword(s):  
Sucrose Solution ◽  
Membrane Integrity ◽  
Dna Integrity ◽  
Ringer’S Solution ◽  
Sperm Dna ◽  
Significant Difference ◽  
Pseudopleuronectes Yokohamae ◽  
Cryopreserved Sperm ◽  
Motility Of Sperm ◽  
Cryopreservation Protocol

The marbled flounder (Pseudopleuronectes yokohamae) is a commercial flatfish in East Asia. The aim of this study was to improve its sperm cryopreservation protocol based on the vitality assessment of 7-day and 1-year cryopreserved sperm. Four extenders (extender-1: sucrose solution; extender-2: glucose solution; extender-3: fish Ringer's solution; and extender-4: modified fish Ringer's solution) were tested with a combination of five cryoprotectants (CPAs) (dimethyl sulfoxide: Me2SO; glycerol: GLY; ethylene glycol: EG; propylene glycol: PG; and methanol: MeOH) at four different concentrations (5, 10, 12, and 15%). Fluorescent technique was applied to detect the plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and DNA integrity of fresh and cryopreserved sperm specimens. Fresh sperm was diluted at a ratio of 1:2 (sperm:extender). Post-thaw motility of sperm cryopreserved using 15% Me2SO along with either extender-1 (86.0 ± 5.2%) or extender-2 (85.7 ± 7.1%) was similar (p > 0.05) to that of fresh sperm. Sperm cryopreserved using 12% GLY combined with extender-1 (83.67 ± 6.7%) or extender-2 (83.3 ± 4.7%) showed a similar motility to those cryopreserved with 15% Me2SO, but significantly lower from fresh sperm. The type of straw (0.25 or 0.50 mL) did not show any significant difference (p > 0.05) in post-thaw sperm motility. The highest values of PMI and MMP were observed for 7-day cryopreserved sperm using extender-1 in combination with 15% Me2SO (91.0 ± 2.9% and 90.0 ± 2.0%, respectively) or 12% GLY (90.0 ± 1.3% and 90.0 ± 4.6%, respectively). These results were similar to those of fresh sperm (95.3 ± 2.1% and 92.9 ± 2.5%, respectively). PMI and MMP of 1-year cryopreserved sperm using extender-1 in combination with 15% Me2SO (90.3 ± 2.5% and 89.3 ± 2.1%, respectively) or 12% GLY (90.0 ± 4.4% and 88.7 ± 2.2%, respectively) were significantly similar (p > 0.05) to those of fresh sperm. Sperm DNA integrity did not reveal any significant difference (p > 0.05) between fresh and cryopreserved (7-day and 1-year) sperm. Based on the assessed sperm vitality indicators, a cryopreservation protocol using extender-1 in combination with 15% Me2SO or 12% GLY has potential for hatchery as well as to create a germplasm bank.

Effects of boar variability on comet-detected sperm-DNA damage following cryopreservation

2018 ◽  
Vol 58 (2) ◽  
pp. 252 ◽  
Author(s):  
L. Fraser ◽  
Ł. Zasiadczyk ◽  
C. S. Pareek
Keyword(s):  
Dna Damage ◽  
Membrane Integrity ◽  
Tail Length ◽  
Dna Integrity ◽  
Comet Tail ◽  
Sperm Dna Damage ◽  
Type Iv ◽  
Sperm Dna ◽  
Damage Type

Assessment of sperm-DNA integrity is a crucial issue in male fertility. In the present study, parameters derived from the image analysis of comets after single-cell gel electrophoresis were used to analyse the types of DNA damage of frozen–thawed boar spermatozoa. Semen, frozen in a cryoprotectant-free extender or in cryoprotectant-based extenders, was analysed for DNA fragmentation and with the following comet tail measures: percentage DNA in comet tail, comet tail length and olive tail moment. The percentages of sperm DNA damage in the comet tails were classified as Type 0 (no DNA damage), Type I (very low DNA damage), Type II (light DNA damage), Type III (medium DNA damage) and Type IV (heavy DNA damage). Sperm motility characteristics and membrane integrity were assessed in the pre-freeze and frozen–thawed semen samples. Assessment of sperm DNA fragmentation and comet tail measures showed marked inter-boar variability following cryopreservation. However, consistent differences among the boars, with respect to cryo-induced sperm DNA damage, were detected by the comet tail length and olive tail moment. Besides Type IV, all types of DNA damage were detected in the cryoprotectant-based extenders. It was found that the frequency of Type II and Type III of DNA damage of frozen–thawed spermatozoa was significantly greater in the cryoprotectant-based and cryoprotectant-free extenders respectively. Deterioration in the quality of the sperm DNA integrity was concomitant with a marked decline in sperm motility characteristics, reduced plasma membrane integrity and higher lipid peroxidation and aspartate aminotransferase activity after cryopreservation. It can be suggested that the comet-assay parameters, coupled with routine laboratory tests, are useful to improve the sperm evaluations of post-thaw quality of semen from individual boars and would offer more comprehensive information for a better understanding of the degree of cryo-induced sperm-DNA damage.

scholarly journals Finding an Effective Freezing Protocol for Turkey Semen: Benefits of Ficoll as Non-Permeant Cryoprotectant and 1:4 as Dilution Rate

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 421 ◽  
Author(s):  
Michele Di Iorio ◽  
Giusy Rusco ◽  
Roberta Iampietro ◽  
Maria Antonietta Colonna ◽  
Luisa Zaniboni ◽  
...  
Keyword(s):  
Dilution Rate ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Dna Integrity ◽  
Osmotic Resistance ◽  
Progressive Motility ◽  
Nitrogen Vapor ◽  
Combined Use ◽  
Cryopreservation Protocol

The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

scholarly journals Effect of cryoprotectant on the motility, viability, fertilization, and DNA integrity of naleh fish Barbonymus sp. (Cyprinidae) sperm

2021 ◽  
Vol 58 ◽  
pp. e168702
Author(s):  
Siti Maulida ◽  
Kartini Eriani ◽  
Firman Muhammad Nur ◽  
Nur Fadli ◽  
Agung Setia Batubara ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Protective Effect ◽  
Egg Yolk ◽  
Dna Integrity ◽  
Similar Concentration ◽  
Breeding Programs ◽  
Ringer’S Solution ◽  
Cryopreserved Sperm ◽  
Cryopreservation Method ◽  
Egg Fertility

Naleh fish Barbonymus sp. is a commercial freshwater fish, which is indigenous to Aceh, Indonesia. The population of this species has declined over the years as a result of habitat perturbations and overfishing. Hence, the crucial need to develop a cryopreservation method to support breeding programs. This involved the use of a cryoprotectant as an important component. The objective of this study, therefore, was to explore the best cryoprotectant for naleh fish spermatozoa, and a total of five types were tested. These include the DMSO, Methanol, Ethanol, Glycerol, and Ethylene Glycol at a similar concentration of 10%, which were individually combined with 15% egg yolk, and every treatment was performed in three replications. Conversely, Ringer’s solution was adopted as an extender, and the sperm was cryopreserved in liquid nitrogen for 15 days. The results showed significant influence on sperm motility and viability, as well as egg fertility of naleh fish (P <0.05), although the DMSO provided the best outcome, compared to others at 47.17%, 50.13%, and 45.67%, respectively. Furthermore, DNA fragmentation had not occurred in the fresh and cryopreserved sperm samples, indicating the protective effect of tested cryoprotectants. It is concluded that the 10% DMSO and 15% egg yolk is the best cryoprotectant for naleh fish spermatozoa.

102 Age-related differences in seminal parameters and expression of fertility marker genes in Marwari stallion

2021 ◽  
Vol 33 (2) ◽  
pp. 158
Author(s):  
A. K. Chaudhary ◽  
G. N. Purohit ◽  
J. S. Mehta ◽  
S. K. Ravi ◽  
T. R. Talluri
Keyword(s):  
Age Groups ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Marker Genes ◽  
Dna Integrity ◽  
Expression Levels ◽  
Seminal Parameters ◽  
Age Related ◽  
Significant Difference ◽  
Artificial Vagina

The objective of the present study was to observe age-related differences in seminal parameters and expression of fertility marker genes in Marwari stallions. Semen was collected from 9 Marwari stallions (6 ejaculates per stallion) of 3 different age groups (2 to 4 years, GI; 4 to 6 years, GII; and &gt;6 years, GIII) twice a week using an artificial vagina by allowing the stallions to mount a mare. The collected ejaculate was divided into 2 parts. Part A was used for evaluation of various seminal parameters (colour, consistency, total volume, gel volume, gel-free volume, pH, progressive sperm motility, sperm concentration, sperm viability, total sperm morphological abnormalities, plasma membrane integrity, acrosomal integrity, and DNA integrity). Part B was centrifuged to obtain the sperm pellet for DNA extraction. Six fertility-related marker genes (SPATA1, SP17, PLCz, CRISP3, UBB, and PRM1) were examined in the spermatozoa DNA using PCR. Expression levels of these genes were also studied using quantitative real-time PCR. Data obtained were analysed statistically by one-way or two-way analysis of variance using the SPSS computer program (version 20.0; IBM Corp.). No significant difference was observed among the GI, GII, and GIII stallions for seminal parameters, except mean sperm concentration, which was lowest in 2- to 4-year-old stallions (146.06±11.50 million mL−1), intermediate in stallions &gt;6 years old (182.03±8.51 million mL−1), and highest in stallions of 4 to 6 years (270.92±9.12 million mL−1; P&lt;0.01). The study demonstrated that all six fertility-related genes showed differential expression in stallions from the GI, GII, and GIII groups. In addition, expression levels of the same genes varied across individuals. Expression of SPATA1, SP17, PLCz, CRISP3, UBB, and PRM1 genes was reduced in stallions below 4 years of age compared with older stallions. This result suggests that expression of these genes increases with age, although possibly only up to a certain age. We inferred that stallions around 4–6 years of age can be considered optimum to use for breeding purposes, provided seminal parameters are normal.

Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome

Reproduction ◽  
2013 ◽  
Vol 146 (5) ◽  
pp. 433-441 ◽  
Author(s):  
Renata Simões ◽  
Weber Beringui Feitosa ◽  
Adriano Felipe Perez Siqueira ◽  
Marcilio Nichi ◽  
Fabíola Freitas Paula-Lopes ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Cleavage Rate ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Significant Difference

Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

scholarly journals Cryopreservation of Aceh Cattle Semen with Date (Phoenix dactylifera) Extract Supplementation

2019 ◽  
Vol 11 (1) ◽  
pp. 117-124 ◽  
Author(s):  
Yonadiah Dwitya ◽  
Kartini Eriani ◽  
Hendra Saputra ◽  
Al-Azhar Al-Azhar ◽  
Muhammad Rizal
Keyword(s):  
Date Palm ◽  
Semen Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Phoenix Dactylifera ◽  
Dna Integrity ◽  
Randomized Design ◽  
Sperm Dna ◽  
Completely Randomized Design ◽  
Fresh Semen

Cryopreservation process could affect spermatozoa quality during from reactive oxygen species (ROS) produced in cellular metabolism and the environment. Spermatozoa damage caused by ROS during cryopreservation can be reduced with the addition of natural antioxidant which commonly found in fruits like date palm. This research was done to investigate the influence of date extract on semen quality after cryopreservation. This experimental study used a completely randomized design with 4 treatments and 6 replications. Semen collected from two aceh cattle bulls was diluted in tris egg yolk extender contained different concentrations (v/v) of date extract: 0% (P0, control), 0.75% (P1), 1% (P2), and 1.25% (P3) before cryopreserved at -196 ºC for 7 days. Semen quality prior to and after cryopreservation as well as sperm DNA integrity were determined by standard microscopic and laddering methods, respectively. The results showed that the addition of 1% date extract could maintain viability (68.67%), plasma membrane integrity (62.33%), and abnormality (18.58%) of aceh cattle spermatozoa, but unable to maintain its motility above 40%. There was no DNA fragmentation observed in both treated and fresh semen. This is the first study investigates the influence of supplementation of date palm extract on preserved aceh cattle spermatozoa diluted in egg yolk tris based extender.

43 CORRELATIONS OF METHODS OF SPERM ANALYSIS IN FRESH SEMEN OF SOUTH AFRICAN INDIGENOUS GOAT

2017 ◽  
Vol 29 (1) ◽  
pp. 129
Author(s):  
O. A. Ajao ◽  
F. Fushai ◽  
D. O. Owiny ◽  
D. M. Barry
Keyword(s):  
Acridine Orange ◽  
Membrane Integrity ◽  
Dna Integrity ◽  
Sperm Analysis ◽  
Sperm Dna ◽  
Water Test ◽  
Sperm Membrane ◽  
Acrosome Integrity ◽  
Live Sperm ◽  
The Individual

This study investigated the correlations between methods of assessing sperm qualities; namely, sperm total motility (TM), sperm vitality, acrosome integrity, DNA integrity, and sperm membrane integrity. A total of 60 ejaculates from 6 bucks were collected and the spermatozoa evaluated. The overall mean percentages of sperm TM, sperm vitality (eosin-nigrosin and propidium iodide stains), sperm acrosome integrity (Spermac and SpermBlue® stains), sperm DNA integrity (acridine orange and halotech), and sperm membrane integrity (hypoosmotic swelling test and water test) were 94.7 ± 0.5, 81.0 ± 0.6. 79.6 ± 3.7, 79.7 ± 1.8, 78.6 ± 5.3, 75.7 ± 5.5, 74.3 ± 5.1, 73.1 ± 3.5, and 73.4 ± 3.6, respectively. There were significant correlations (P < 0.05) between mean percentage live sperm evaluated with eosin-nigrosin stain and sperm TM (r = 0.813), between percentage intact acrosome assessed with SpermBlue® and sperm TM (r = 0.846), and between SpermBlue® and eosin-nigrosin (r = 0.965). There were highly significant correlations (P < 0.01) between sperm membrane integrity evaluated with HOS test and sperm TM (r = 0.871), between percentage of intact sperm DNA assessed with halotech and SpermBlue® (r = 0.832), and between percentage of intact spermatozoa DNA assessed with acridine orange and percentage intact acrosome evaluated with spermac stain (r = 0.862). Under the conditions of this study, the correlated methods of sperm analysis proved suitable for analysis of goat spermatozoa and can serve as useful indicator of potential fertility for sperm. They could be used for accurate assessment of the individual sperm cell rather the population as a whole. Motility, eosin-nigrosin stain, SpermBlue®, halotech and acridine orange stain still remain practical and valuable tools for predicting sperm fertilizing ability.

26 Baobab oil supplemented extender preserves post-thaw bull sperm quality parameters

2020 ◽  
Vol 32 (2) ◽  
pp. 139
Author(s):  
Z. Raphalalani ◽  
F. Ramukhithi ◽  
R. Ndhlala ◽  
K. Nephawe ◽  
T. Nedambale
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Control Group ◽  
Dna Integrity ◽  
Sperm Dna ◽  
Frozen Semen ◽  
Morphological Defects ◽  
Bull Sperm ◽  
Semen Extender

The processes of semen cryopreservation and thawing affect sperm membrane integrity and motility and increases morphological defects as well as DNA damage. The most influential cause of this is oxidative stress. When endogenous antioxidant capacity of seminal plasma is reduced during the freeze-thawing process, plant extracts exhibiting strong antioxidant activity can be used as supplements for compensation. Baobab oil has gained interest because it is rich in powerful antioxidants, which could protect sperm cells from oxidative damage during cryopreservation. Our study aimed to assess the effects of baobab oil on post-thaw sperm quality parameters in an egg-yolk-based extender. Thirty semen ejaculates were collected from 15 Nguni bulls using an electro ejaculator. Semen samples were randomly allocated to control (no baobab oil), 20μL (1%), 50μL (2.5%), and 100μL (5%) baobab oil per millilitre extender. Following dilution, semen samples were loaded into 0.25-mL semen straws, equilibrated for 4h at 5°C, and transferred into a controlled rate programmable freezer. The frozen semen straws were stored in a liquid nitrogen tank (−196°C) until thawing. Semen straws were thawed (37°C/60 minutes) after 1 week of cryopreservation and analysed for (1) sperm motility using a computer-aided sperm analyser, (2) morphological defects and viability using eosin-nigrosin stain, (3) membrane integrity by hypo-osmotic swelling test, and (4) DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Data was analysed using analysis of variance. Treatment means were compared in relation to the control group by Dunnett's test. We found that supplementing semen extender with baobab oil at 1% significantly (P&lt;0.05) preserved sperm DNA integrity (88.3±3.7) and membrane integrity (74.0±4.2) when compared with the control group (71.7±3.7 and 55.8±4.4, respectively). Baobab oil supplementation either at 1% (5.9±0.5), 2.5% (7.2±0.5), or 5% (6.0±0.5) significantly reduced sperm morphological defects compared with control (9.5±0.5). Total motility (1% (72.7%), 2.5% (72.7%), 5% (71.9%), control (59.3%)) and viability (1% (79.1%), 2.5% (79.8%), 5% (77.8%), control (67.6%)) were also improved by supplementation; however, the difference was not significant. In conclusion, it was demonstrated that supplementing bull semen extender with 1% baobab oil protects sperm from morphological defects, maintains membrane integrity, as well as preserves sperm DNA. All the baobab oil supplementation levels preserved post-thaw bull-sperm quality parameters.

scholarly journals Cryopreservation of spermatozoa of indigenous stinging catfish, Heteropneustes fossilis (Bloch) for ex-situ conservation

2020 ◽  
Vol 32 (1) ◽  
pp. 11-22
Author(s):  
MD. RAFIQUL ISLAM SARDER ◽  
MD. ABUL KALAM AZAD ◽  
K.M. SHAHRIAR NAZRUL ◽  
MOHAMMAD RASHED
Keyword(s):  
Ex Situ Conservation ◽  
Egg Yolk ◽  
Ringer Solution ◽  
The Other ◽  
Ex Situ ◽  
Nacl Solution ◽  
Heteropneustes Fossilis ◽  
Significant Difference ◽  
Cryopreserved Sperm ◽  
Cryopreservation Protocol

An experiment was conducted to develop cryopreservation protocol for spermatozoa of stinging catfish, Heteropneustes fossilis and to use the cryopreserved sperm in its breeding trials. The activation of sperm motility at various concentrations of NaCl solution was tested and complete activation and inhibition of sperm were obtained at 0.4% and 0.9% to 1% NaCl solution respectively. In toxicity test, sperms were incubated with DMSO, methanol and ethanol at 5, 10, and 15% concentrations where DMSO and methanol produced better motility at 5 and 10% concentration with Alsever’s solution and egg-yolk citrate at 5 and- 10 min incubation period. Three extenders- Alsever’s solution, egg-yolk citrate and Ginsburg Fish Ringer solution and three cryoprotectants- DMSO, methanol and ethanol were used for cryopreservation of sperm, and among the diluents, Alsever’s solution with 10% DMSO showed best performance producing 77.50±3.22% post-thaw motility. On the other hand, egg-yolk citrate and Ginsburg Fish Ringer solution along with 10% DMSO produced 63.75±2.39% and 62.50±3.22% post-thaw motility, respectively. Sperm preserved with Alsever’s solution plus 10% DMSO produced 52.5±3.34% and 38.0±2.39%fertilization and hatching, and those preserved with Alsever’s solution plus 10% methanol produced 46.9±3.11% and 32.7±2.70% fertilization and hatching respectively. The fry produced using cryopreserved and fresh sperm grew well and no significant difference (p>0.05) was found between two groups.

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