Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm

2016 ◽  
Vol 52 (2) ◽  
pp. 257-263 ◽  
Author(s):  
M Nichi ◽  
T Rijsselaere ◽  
JDA Losano ◽  
DSR Angrimani ◽  
GKV Kawai ◽  
...  
Keyword(s):  
Membrane Potential ◽  
In Vitro Fertilization ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Storage Temperature ◽  
Membrane Integrity ◽  
Epididymal Sperm ◽  
Vitro Fertilization

scholarly journals D-Chiro-Inositol Improves Sperm Mitochondrial Membrane Potential: In Vitro Evidence

2020 ◽  
Vol 9 (5) ◽  
pp. 1373 ◽  
Author(s):  
Rosita A. Condorelli ◽  
Federica Barbagallo ◽  
Aldo E. Calogero ◽  
Rossella Cannarella ◽  
Andrea Crafa ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Polycystic Ovarian Syndrome ◽  
Biologically Active ◽  
Ovarian Syndrome

The use of inositols in endocrinological clinical practice is increasingly widespread. Most of the existing evidence concerns myoinositol (MYO), the most abundant form in nature, especially in women with polycystic ovarian syndrome. We have previously shown that MYO increases sperm motility in patients with asthenozoospermia by the increase of sperm mitochondrial membrane potential (MMP), a biofunctional sperm parameter closely associated to sperm motility. The aim of this study was to evaluate the effects of D-chiro-inositol (DCI), another biologically active isoform of inositols, on sperm MMP, as data on this matter has never been released so far. To accomplish this, semen samples from 15 patients with asthenozoospermia and 15 healthy normozoospermic men were incubated with increasing concentrations of DCI (0, 75, and 750 µg/mL) or phosphate buffer saline for 30 min. Incubation with DCI significantly improved sperm MMP at lower concentrations, and with shorter incubation length than those used in our similar MYO studies. In conclusion, these findings indicate that DCI positively impacts on sperm mitochondrial function in vitro. Studies aimed at assessing the role of DCI in the treatment of asthenozoospermia in-vivo are warranted.

scholarly journals Outlining adequate protocols for Lidia bull epididymal storage and sperm cryopreservation: use of glycerol, dimethylformamide and N-acetylcysteine

2017 ◽  
Vol 15 (3) ◽  
pp. e0405
Author(s):  
Elvira Matilla ◽  
Lauro González-Fernández ◽  
Felipe Martínez-Pastor ◽  
Nuria Hernández ◽  
Carolina Tobajas ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Egg Yolk ◽  
Ros Production ◽  
Epididymal Sperm ◽  
Sperm Cryopreservation ◽  
Cattle Industry ◽  
Bull Sperm

The Lidia bovine breed is an important hallmark of the Spanish cattle industry. Bulls are selected based upon aggressiveness and epididymal sperm cryopreservation is the way to obtain and store their genetics. There are not specifically designed protocols yet to perform Lidia bull sperm cryopreservation. The present study aimed to determine if a tris-fructose-citrate-egg yolk (20% v/v; TFY) extender supplemented with 7% glycerol (TFY1) or 3.5% glycerol plus 3.5% dimethylformamide (DMF; TFY2) are suitable media for cryopreservation of epididymal Lidia bull sperm. Moreover, the effect of N-acetylcysteine (NAC), a potent antioxidant, was evaluated. The epididymis were stored at 4°C for 24, 48, 72 or 96 h, and both freezing media were tested as such or supplemented with 1 or 2.5 mM of NAC. Our data demonstrated that post-thaw viability was well maintained (TFY1: 50.8% ± 1.9 at 24 h and 52.4% ± 0.8 at 96 h and TFY2: 52.6% ± 1.6 at 24 h and 56.1% ± 1.8 at 96 h; mean % ± SEM; p>0.05) as also were total and progressive sperm motility, high mitochondrial membrane potential, ROS production, DNA status and acrosomal intactness of Lidia bull sperm up to 96 h of epididymal storage, all extender variations being similar (p>0.05). In conclusion, the use of TFY medium supplemented either with 7% glycerol alone or the combination of 3.5% glycerol and 3.5% DMF were equally safe choices for epididymal Lidia bull sperm cryopreservation, and NAC addition did not significantly improve sperm post-thaw quality.

scholarly journals Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3309
Author(s):  
Michal Ďuračka ◽  
Kamila Husarčíková ◽  
Mikuláš Jančov ◽  
Lucia Galovičová ◽  
Miroslava Kačániová ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Dna Fragmentation ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Control Group ◽  
Sperm Dna Fragmentation ◽  
Bovine Semen ◽  
Sperm Dna

Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The Staphylococcus genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of Staphylococcus-induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll® Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven Staphylococcus species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 108 colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of S. aureus, S. warneri, S. kloosii, and S. cohnii caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of Staphylococcus species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of Staphylococcus bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

Ram spermatozoa migrating through artificial mucus in vitro have reduced mitochondrial membrane potential but retain their viability

2015 ◽  
Vol 27 (5) ◽  
pp. 852 ◽  
Author(s):  
Carmen Martínez-Rodríguez ◽  
Mercedes Alvarez ◽  
Elena López-Urueña ◽  
Susana Gomes-Alves ◽  
Luis Anel-López ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Mucus Penetration ◽  
Sperm Counts ◽  
Sample Test

Sperm motility in vitro is one of the most common predictors of fertility in male screening. We propose that a mucus-penetration assay can isolate a cellular subpopulation critical to reproductive success. To this end, a device was designed with three modules (sample, test and collection) and its conditions of use evaluated (length of mucus, incubation time, mucus medium, sperm concentration and position in relation to the horizontal). The number of spermatozoa migrating and the viability and acrosomal status of the spermatozoa not migrating were calculated. The second objective was to evaluate the qualitative parameters of the spermatozoa migrating in 1.6% polyacrylamide for 30 min. The number of spermatozoa migrating and the sperm motility, viability and the acrosomal and mitochondrial status of three sperm populations (fresh, not migrating and migrating) were determined. A higher number of migrating spermatozoa were observed after 60 min of incubation, but this situation adversely affected sperm quality. The methylcellulose-based test showed a significantly lower number of migrating spermatozoa than the polyacrylamide test. The position at an angle of 45° resulted in a higher number of migrating spermatozoa in the polyacrylamide-based test. The sperm counts for three consecutive assays indicated an acceptable repeatability of the method. The viability and acrosomal status of the migrating spermatozoa showed no significant changes with regard to the control when the device was placed at 45°, whereas these parameters showed lower values at 0°. The percentage of high mitochondrial membrane potential spermatozoa was significantly reduced in the population of migrating spermatozoa.

297 BIRTH OF LIVE RATS THROUGH IN VITRO FERTILIZATION USING CRYOPRESERVED SPERMATOZOA: SPERM FERTILITY IMPROVED BY FREEZING AT - 150°C

2006 ◽  
Vol 18 (2) ◽  
pp. 256
Author(s):  
Y. Seita ◽  
Y. Okuda ◽  
A. Takizawa ◽  
S. Hisamatu ◽  
T. Inomata ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Plasma Membrane Integrity ◽  
Freezing Temperatures

We previously reported that damages to spermatozoa by cold shock can be avoided by cooling slowly at 0.5�C/min to 5.0�C (Seita et al. 2005 Reprod. Fertil. Dev. 17, 277-278). The objective of the present study was to develop an in vitro fertilization (IVF) system with frozen-thawed rat spermatozoa for more efficient reproduction of live offspring. We examined the effect of freezing temperatures (cooling 5.0�C to pre-plunging) on post-thaw sperm motility, plasma membrane integrity, and fertility in vitro. Epididymal spermatozoa of Wistar rats were collected in 2.0 mL of freezing medium containing 23% (v/v) egg yolk, 8.0% (w/v) lactose monohydrate, and 0.7% (v/v) Equex STM (Nova Animal Sales, Inc., Scituate, MA, USA) at room temperature. Samples were loaded into 0.25-mL straws and cooled to 5.0�C at 0.5�C/min in a programmable freezer. Next, the samples were exposed to liquid nitrogen (LN) vapor at various freezing temperatures (-120�C, -150�C or -180�C) above the LN level for 15 min and then plunged into LN. Straws were thawed in a 37�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL in a droplet of 200 �L of R1ECM and then pre-incubated for 5 h. Ovulated oocytes were introduced into the droplet and co-cultured for 10 h. The oocytes were denuded, fixed, and/or examined for two pronuclei (2PN) formation microscopically. The denuded oocytes, which were fertilized with spermatozoa frozen at -150�C and were microscopically confirmed to have 2PN formation, were transferred to pseudo-pregnant recipient females. IVF was also performed by the same method using fresh spermatozoa as the control. Differences in the sperm motility and plasma membrane integrity were analyzed by ANOVA, and the IVF data were analyzed by chi-square test. At 2 h after thawing the motility of spermatozoa frozen at -150�C was significantly higher than that of spermatozoa frozen at -180�C (19.8% and 11.1%; P < 0.05), although the sperm plasma membrane integrity was not significantly different among different freezing temperatures, -120�C, -150�C, and -180�C (18.2%, 23.5%, and 17.9%; P > 0.05). The percentage of oocytes with 2PN was not significantly different between the -150�C frozen and the control (fresh spermatozoa) groups [59% (131/221) and 62% (155/251); P > 0.05], although that of frozen spermatozoa at -120�C and -180�C [20% (38/188) and 23% (35/153)] were significantly lower than that of frozen spermatozoa at -150�C (P < 0.05). A total of 168 putative fertilized zygotes with 2PN were transferred to eleven recipients, and 87 live young were born. In conclusion, our results indicated that post-thaw motility of cryopreserved rat spermatozoa was improved by using a suitable cooling protocol, and the IVF system used in the present study would effectively produce offspring from the cryopreserved epididymal rat spermatozoa. To our knowledge, this procedure is the first successful production of live offspring from cryopreserved rat spermatozoa through in vitro fertilization.

168 IMPROVED MITOCHONDRIAL MEMBRANE POTENTIAL AND PRE-IMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS BY DIBUTYRYL cAMP TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 192 ◽  
Author(s):  
J.-S. Kim ◽  
D.-B. Koo ◽  
B.-S. Song ◽  
G. B. Wee ◽  
K.-K. Lee ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Embryonic Development ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Developmental Stages ◽  
Treated Group ◽  
Dibutyryl Camp ◽  
Germinal Vesicle Breakdown ◽  
Vitro Fertilization

Mitochondria play a pivotal role in energy metabolism and apoptosis during embryo development. In general, cAMP that exists at high level in GV oocytes inhibits germinal vesicle breakdown (GVBD), and the amount of cAMP in oocyte cytoplasm is gradually decreased for meiotic resumption. We first examined the effects of dibutyryl cAMP (dbcAMP) on nuclear maturation, fertilization, and early embryonic development. To determine whether mitocondrial activity is related to embryonic development, mitochondrial membrane potential (ΔΨm) was measured in porcine embryos. Porcine oocytes were cultured in NCSU-23 medium supplemented with 1 mM dbcAMP for 22 h and further cultured without dbcAMP for 22 h. After in vitro fertilization, porcine eggs were cultured in NCSU-23 medium with 4% BSA at 39�C, 5% CO2 in air for 6 d. Porcine embryos were obtained at various developmental stages and stained with the mitochondrial membrane potential-sensitive dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide). The mitochondrial membrane potential of porcine embryos was quantitatively evaluated by the ratio of green (presumptively alive mitochondria) to red fluorescent signal (presumptively dead mitochondria) using a fluorescence microscope. Acquired images were analyzed using DeltaVision Software (Applied Precision, Inc., Hsin-Chu City, Taiwan, ROC). After completion of IVM, dbcAMP-treated oocytes (91.3 � 0.9%) showed a higher proportion of the metaphase II stage than nontreated ones (72.8 � 2.6%) (P < 0.05). In the dbcAMP-treated group, sperm penetration rate was increased and polyspermic rate was reduced as compared to the nontreated group. Furthermore, the rate (37.3%, 47/126) of blastocyst formation in dbcAMP-treated group was higher than that (19.2%, 28/146) of the nontreated group (P < 0.05). After JC-1 staining, the number of blastomeres having live mitochondria per embryo increased in the dbcAMP-treated group at various developmental stages, whereas the number of blastomeres having dead mitochondria per embryo was enhanced in the nontreated group (43.3% vs. 30.2%). Our results indicate that in vitro maturation of porcine oocytes may affect mitochondrial membrane potential, apoptosis, and embryonic quality during pre-implantation development.

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