scholarly journals 202FERTILIZING ABILITY OF EPIDIDYMAL CAT SPERMATOZOA AFTER CRYOPRESERVATION OR STORAGE AT 4°C.

2004 ◽  
Vol 16 (2) ◽  
pp. 222 ◽  
Author(s):  
L. Bogliolo ◽  
P. Bonelli ◽  
L. Madau ◽  
M.T. Zedda ◽  
C. Santucciu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Genetic Material ◽  
Membrane Integrity ◽  
Long Distance ◽  
Vitro Fertilization ◽  
Plasma Membrane Integrity ◽  
Fertilization Rates ◽  
Epididymal Spermatozoa

The possibility of harvesting cat epididymal spermatozoa from excised testis represents a potentially important tool for preserving valuable genetic material from males that die unexpectedly. The purpose of this study was to evaluate plasma membrane integrity, acrosomal status and in vitro fertilizing capability of epididymal cat sperm after short- and long-term storage at 4°C and after cryopreservation. Spermatozoa were collected by flushing the cauda epididymis and the vasa deferentia removed from adult cats undergoing orchiectomy. Spermatozoa were split into three aliquots: A-fresh, B-frozen, and C-cooled. The A portion was immediately tested on the day of collection (control), the B portion was frozen in pellets in Test Yolk Buffer (TYB) with 8% glycerol, while the C portion was extended in TYB and stored at 4°C for 1 day, 2 days or 7 days. Cat oocytes recovered from minced ovaries were matured for 24h in TCM 199+1UI/mL hCG+0.5UI/mL FSH+0.3% BSA at 38.5°C in 5% CO2. In vitro fertilization (IVF) of in vitro-matured (IVM) oocytes was performed using spermatozoa from portions A, B or C in modified Tyrode’s solution supplemented with 0.6% BSA at 38.5°C in 5% in air. Before IVF, aliquots of sperm samples from the three portions were stained simultaneously with fluorescein isothiocyanate-labelled Pisum Sativum agglutinin (FITC-PSA) and propidium iodide to evaluate the percentage of plasma membrane-intact spermatozoa with acrosomes present. At least 200 spermatozoa were counted in duplicate for each sample. After 24h of incubation, to evaluate fertilization rate, the oocytes were stained with aceto-lacmoid. Oocytes with two pronuclei, presumably male and female, were classified as fertilized. The percentages of spermatozoa maintaining plasma membrane integrity and intact acrosomes after 1 day or 2 days of storage at 4°C were 75% and 65%, respectively, and were similar to that of fresh epididymal spermatozoa (78%). However, the percentages of spermatozoa with intact plasma membranes that had intact acrosomes after 7 days of storage at 4°C (52%) or after cryopreservation (48%) were significantly lower (P<0.01, ANOVA) than those of samples stored for 1 or 2 days at 4°C. As shown in the Table, fertilization rates of oocytes inseminated with fresh spermatozoa and spermatozoa stored for 1 or 2 days were similar. In contrast, the fertilization rates of oocytes inseminated with spermatozoa that had been cryopreserved or stored for 7 days were significantly lower than those obtained with spermatozoa used immediately after collection or storage for 1 day. From our results, we suggest that storage of epididymal spermatozoa at 4°C may be a potentially useful method for temporary storage of spermatozoa from endangered cats, and may allow more efficient use and long-distance transport of genetically important germplasm. This work was supported by MIUR (ex 40%). Table 1 In vitro fertilization of in vitro-matured cat oocytes with fresh, stored at 4°C and cryopreserved epididymal cat 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.

scholarly journals In Vitro Fertilization Outcome After Intracytoplasmic Sperm Injection with Fresh and with Frozen-Thawed Epididymal Spermatozoa

KnE Medicine ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Hilma Putri Lubis
Keyword(s):  
In Vitro Fertilization ◽  
Intracytoplasmic Sperm Injection ◽  
Oocyte Retrieval ◽  
Epididymal Sperm ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Sperm Aspiration ◽  
Significant Difference ◽  
Epididymal Spermatozoa

<p><strong>Introduction</strong></p><p>Testicular epididymal sperm aspiration (TESA) is one of the method  to retrieve sperm from the testes in men with azoospermia. The aim of the study is to compare the In vitro fertilization (IVF) outcome of intracytoplasmic sperm injection (ICSI)-ET cycles with fresh testicular epididymal spermatozoa obtained on the same day with  oocyte retrieval and with frozen-thawed testicular epididymal spermatozoa.</p><p><strong>Material &amp; Methods</strong></p><p>A retrospective comparative analysis of  patients who underwent fresh TESA and frozen-thawed TESA in ICSI-ET cycles from January 2012 to December 2014 in Halim Fertility Center was done. Fresh testicular epididymal sperm aspiration (fresh TESA) was performed on the same day with oocyte retrieval in 28 cycles and the frozen-thawed testicular epididymal sperm aspiration (frozen-thawed TESA) was used in 30 cycles.  </p><p><strong>Results</strong></p><p>The two groups were comparable in terms of the ages of male and female patients, etiology of infertility and duration of infertility. Fertilization rates in fresh TESA group were 53,5% and in frozen-thawed TESA group, fertilization rates were 50%. There was no statistically significant difference between the groups. Clinical pregnancy rates in fresh TESA group were 35,7%  and in frozen-thawed TESA group, clinical pregnancy rates were 26,7% and statistically there was no significant difference between the groups.</p><p><strong>Conclusion</strong></p>There is no significant difference in the in vitro fertilization outcome of intracytoplasmic sperm injection (ICSI)-ET cycles between fresh TESA and frozen-thawed TESA .

161 IN VITRO FERTILIZATION OF DROMEDARY CAMEL (CAMELUS DROMEDARIUS) OOCYTES WITH EPIDIDYMAL SPERMATOZOA

2012 ◽  
Vol 24 (1) ◽  
pp. 192 ◽  
Author(s):  
A. R. Moawad ◽  
G. M. Darwish ◽  
M. R. Badr ◽  
A. B. El-Wishy
Keyword(s):  
In Vitro Fertilization ◽  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Dromedary Camel ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Significant Difference ◽  
Epididymal Spermatozoa ◽  
Normal Fertilization

Various techniques such as AI and ET have been reported to improve reproductive efficiency and genetic potential in camelids. In vitro fertilization and the development of IVP embryos are considered an alternative for genetic improvement in this species. This study investigated the effects of different sperm cell concentrations (1, 2, 3 and 4 × 106 sperm mL–1), different capacitating materials (5 mM caffeine, 10 μg mL–1 of heparin, 10 mg mL–1 of theophylline, 1 mM calcium ionophore A23178 and 10 μg of heparin + 5 mM caffeine), post-slaughter epididymal flushing time and fertilization media supplements (Fert-TALP + 6 mg mL–1 of BSA and Fert-TALP + 3 mg mL–1 of polyvinylpyrrolidone ) on fertilization rates and subsequent development of dromedary camel oocytes. Cumulus–oocyte complexes obtained at slaughter were matured in vitro in TCM-199 for 36 h at 39°C in a humidified atmosphere of 5% CO2. For IVF, spermatozoa were collected from epididymides of slaughtered male camels at 1 to 2 h post-slaughter or after 24 h of epididymal storage at 4°C. The spermatozoa were then prepared for IVF by the swim-up technique. Following sperm capacitation, oocytes and spermatozoa were co-incubated for 18 h. Oocytes were then stained using aceto-orcein for evaluation of fertilization events. Presumptive zygotes were cultured in vitro in TCM-199 medium supplemented with 5% FCS for 9 days at 39°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At least 3 replicates were performed for each experimental group. Data were analysed by chi-square test. Fertilization rates were 55.5, 62.5, 62.7 and 47.2% in oocytes inseminated with 1, 2, 3, or 4 × 106 sperm mL–1, respectively. Normal fertilization rate (oocytes with 2 pronuclei) was higher (P =  0.06) in oocytes inseminated with 2 × 106 sperm mL–1 (29.7%) than in those inseminated by 4 × 106 sperm mL–1 (11.1%). Treatment of epididymal spermatozoa with 5 mM caffeine significantly increased (P ≤ 0.05) fertilization rate (61.9%) compared with calcium ionophore A23178 (32.4%). These values were not significantly different from other groups (38.5, 54.1 and 50.0% in heparin, theophylline and heparin + caffeine, respectively). Normal fertilization was highest (25.4%) in oocytes inseminated with caffeine-treated spermatozoa. Insemination of oocytes in Fert-TALP medium containing BSA resulted in a higher fertilization rate (21.4%) compared with oocytes in polyvinylpyrrolidone-supplemented medium (5.7%; P =  0.06). Storage of camel epididymides at 4°C for 24 h did not affect fertilization rates. Cleavage rate (48 h post-insemination) was higher in oocytes fertilized with caffeine-treated spermatozoa than in oocytes in the theophylline group (26.8 vs 10.5%; P =  0.08). No significant difference was observed in the frequency of blastocyst development (5 days post-insemination) between the 2 groups (5.4 vs 2.6%); based on the number of cleaved oocytes, the same proportions of blastocyst embryos were reported (20.0 and 25.0%). Taken together, these results suggest that dromedary camel oocytes can be matured, fertilized and subsequently developed in vitro with high developmental potential. Epididymal spermatozoa at a concentration of 2 × 106 sperm mL–1 prepared in a medium containing caffeine as a capacitating agent can be used effectively in IVF of camel oocytes.

312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION

2007 ◽  
Vol 19 (1) ◽  
pp. 272
Author(s):  
N. Satake ◽  
A. K. Alhaider ◽  
W. V. Holt ◽  
P. F. Watson
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Plasma Membranes ◽  
Fertilization Rate ◽  
Apical Plasma Membrane ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fertilization Rates

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P &lt; 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P &lt; 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.

scholarly journals Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

2015 ◽  
Vol 29 (5) ◽  
pp. 646-651 ◽  
Author(s):  
N. Prapaiwan ◽  
T. Tharasanit ◽  
S. Punjachaipornpol ◽  
D. Yamtang ◽  
A. Roongsitthichai ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Low Density Lipoprotein ◽  
Membrane Integrity ◽  
Density Lipoprotein ◽  
Low Density ◽  
Plasma Membrane Integrity ◽  
Epididymal Spermatozoa

scholarly journals In vitro fertilizing potential of urethral and epididymal spermatozoa collected from domestic cats (Felis catus)

2017 ◽  
Vol 20 (1) ◽  
pp. 19-24 ◽  
Author(s):  
S. Prochowska ◽  
W. Niżański
Keyword(s):  
Comparative Analysis ◽  
In Vitro Fertilization ◽  
Felis Catus ◽  
Domestic Cats ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Commercial Medium ◽  
Blastocyst Rate ◽  
Epididymal Spermatozoa

Abstract The aim of this study was to provide a comparative analysis of in vitro fertilizing potential of frozen-thawed urethral and epididymal feline spermatozoa. Both types of semen were collected from 7 cats and cryopreserved in liquid nitrogen. To perform in vitro fertilization, both urethral and epididymal samples from the same individual were thawed and spermatozoa were co-incubated with in vitro matured cat oocytes. Obtained embryos were cultured in vitro for 7 days in a commercial medium. Cleavage rate, morula rate and blastocyst rate were calculated. Experiment was run in 10 replicates. The examined parameters showed no significant differences between urethral and epididymal spermatozoa (p>0.05). Cleavage rate and embryo’s development were highly variable between replicates, even for the different sperm samples collected from one individual. There was no significant correlation between fertilizing capacity of two types of spermatozoa collected from the same male. In this study we confirmed that cryopreserved urethral spermatozoa have equally good fertilizing potential as epididymal ones, and both can be successfully used for in vitro fertilization in cats with the use of commercial medium.

173 Assessment of the first polar body quality and viability in bovine

2019 ◽  
Vol 31 (1) ◽  
pp. 211
Author(s):  
O. Briski ◽  
M. Duque-Rodríguez ◽  
A. Gambini ◽  
N. P. Leopardo ◽  
E. A. Ynsaurralde ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Dna Fragmentation ◽  
Polar Body ◽  
Genetic Material ◽  
Membrane Integrity ◽  
Genetic Diagnosis ◽  
Mature Oocyte ◽  
Plasma Membrane Integrity ◽  
Apoptotic Process ◽  
First Polar Body

In female gametes, after the first asymmetric meiotic division, a mature oocyte in metaphase II and a first polar body (PB1) are generated. The PB1 contains one of each pair of homologous chromosomes present in the mature oocyte and its DNA can be used for preconception genetic diagnosis. The PB1 degenerates shortly after extrusion, possibly due to an apoptotic process; however, it has not yet been elucidated in bovine. Therefore, the objective of this study was to evaluate PB1 morphology changes, plasma membrane integrity, and the presence of DNA fragmentation during in vitro maturation (IVM). To this aim, cumulus-oocyte complexes collected from slaughterhouse ovaries were cultured in maturation medium in different groups according to IVM time: 16, 18, 20, 22, 24, and 26h. The PB1 were classified into 5 categories according to their morphology: grade (G)1, round PB1 with intact smooth membrane; G2, round or ovoid PB1 with intact membrane; G3, broken PB1 with a small PB1 fragment; G4, broken PB1 with a big PB1 fragment; and G5, completely damaged PB1. Grades 1 and 2 were considered good quality. Plasma membrane integrity was assessed by propidium iodide (PI) DNA staining; PI is a fluorescent intercalating agent that cannot cross the membrane of live cells. The presence of DNA fragmentation was detected at 16, 22, and 26h by TUNEL assay. Data were analysed by two-way ANOVA and Bonferroni post hoc test using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA) and differences were considered significant at P &lt; 0.05. Our results (mean%±s.e.m.) showed that significantly more oocytes assessed at 18, 20, 22, and 24h after onset of IVM presented high-quality (G1) PB1 (18 h: 61.5±13.4%, 20 h: 73.5±9.6%, 22 h: 61.0±9.5%, 24 h: 60±5.1%), compared with those assessed at 16 and 26h (43.0±4.7%, 22.3±3.4%, respectively). The percentage of G2 PB1 did not change throughout the period studied (16 h: 34.0±13.5%, 18 h: 29.9±14.1%, 20 h: 22.0±7.1%, 22 h: 26.5±4.2%, 24 h: 23.3±4.9%, 26 h: 21.33±9.9%), but was significantly lower than that of G1 PB1 at 20, 22, and 24h. The proportion of damaged (G5) PB1 started to increase at 24h (14.3±8.6%), being highest at 26h (30.0±10.5%), in parallel with positive PI staining (P&lt;0.05). Moreover, there was a significant increase of PB1 with DNA fragmentation at 26h (82.0±18.0%) compared with 16h (13.9±9.0%) and 22h (2.5±2.5%). Altogether, these findings demonstrate that PB1 remains stable and of good quality between 18 and 24 h; however, after this time, plasma membrane integrity is compromised and the DNA is fragmented, suggesting the occurrence of an apoptotic process. Our results could be helpful to determine the optimal time for using PB1 as a potential donor of genetic material.

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