scholarly journals Replacement of Albumin by Preovulatory Oviductal Fluid in Swim-Up Sperm Preparation Method Modifies Boar Sperm Parameters and Improves In Vitro Penetration of Oocytes

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1202
Author(s):  
Sergio Navarro-Serna ◽  
Evelyne París-Oller ◽  
Ondrej Simonik ◽  
Raquel Romar ◽  
Joaquín Gadea
Keyword(s):  
Calcium Concentration ◽  
Preparation Method ◽  
Culture Media ◽  
Penetration Rate ◽  
Sperm Parameters ◽  
High Concentration ◽  
Sperm Preparation ◽  
Boar Sperm ◽  
Oviductal Fluid

More suitable and efficient methods to protect gametes from external harmful effects during in vitro handling can be achieved by adding preovulatory porcine oviductal fluid (pOF) to in vitro culture media. The objective of this study was to assess the swim-up procedure’s suitability as a sperm selection method using a medium supplemented with 1mg/mL BSA, 1% preovulatory pOF (v/v), 1% v/v pOF plus 1mg/mL BSA, and 5mg/mL BSA. After selection, various sperm parameters were studied, such as sperm recovery rate, sperm morphology, motility (by CASA), vitality, acrosome status and intracellular calcium (by flow cytometry) and ability to penetrate oocytes in vitro. Around 2% of sperm were recovered after swim-up, and the replacement of BSA by pOF showed a beneficial reduction of motility parameters calcium concentration, resulting in an increased penetration rate. The combination of albumin and oviductal fluid in the medium did not improve the sperm parameters results, whereas a high concentration of BSA increased sperm morphological abnormalities, motility, and acrosome damage, with a reduction of calcium concentration and penetration rate. In conclusion, the replacement of albumin by preovulatory oviductal fluid in the swim-up sperm preparation method modifies boar sperm parameters and improves the in vitro penetration of oocytes.

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
D. B. Koo ◽  
J. S. Park ◽  
K. K. Lee ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Developmental Rate ◽  
Treated Group ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Frozen Semen ◽  
Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

Development of prepubertal goat oocytes after their in vitro maturation and chemical activation

Zygote ◽  
2020 ◽  
Vol 28 (6) ◽  
pp. 447-452
Author(s):  
Seungbum Hong ◽  
Binoy S. Vettical ◽  
Nisar Ahmad Wani
Keyword(s):  
Culture Media ◽  
Chemical Activation ◽  
Control Treatment ◽  
Developmental Potential ◽  
Embryo Culture Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Further Development ◽  
Oviductal Fluid

SummaryExperiments were conducted to study in vitro maturation of prepubertal goat oocytes and their developmental potential after chemical activation. In Experiment 1, cumulus–oocytes complexes collected from the ovaries of prepubertal goats slaughtered at a local abattoir were matured in vitro in TCM-199-based medium supplemented with 10 µg/ml luteinizing hormone (LH) (treatment 1) or 10 µg/ml LH + 0.1 mM l-cysteine (treatment 2). In Experiment 2, mature oocytes were activated with either 5 µM ionomycin or 7% ethanol. After 18 h, some oocytes were randomly fixed and stained to evaluate their chromatin status, while others were cultured in embryo culture medium to study their further development. In Experiment 3, oocytes activated with 5 µM ionomycin were cultured for 7 days in one of the four different culture media [Charles Rosenkrans medium (CR-1), TCM-199, potassium simplex optimization medium (KSOM) and synthetic oviductal fluid (SOF)] to study their developmental potential. The maturation rate in control, treatment 1, and treatment 2 media did not differ from each other (P > 0.05). However, the lowest degeneration of oocytes was observed in treatment 3 (P < 0.05) when compared with the other two groups. The proportion of activated oocytes was higher, while non-activated oocytes were lower in ionomycin group when compared with the group activated with ethanol (P < 0.05). The proportions of oocytes cleaved were 65.7, 56.8, 61.0 and 54.4% in CR-1, TCM-199, KSOM and SOF medium, respectively, with no significant difference. However, further development of cleaved oocytes was better in KSOM followed by SOF.

scholarly journals Selection of Boar Sperm by Reproductive Biofluids as Chemoattractants

Animals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 53
Author(s):  
Luis Alberto Vieira ◽  
Alessia Diana ◽  
Cristina Soriano-Úbeda ◽  
Carmen Matás
Keyword(s):  
Calcium Channel ◽  
Conditioned Medium ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Boar Sperm ◽  
Oviductal Fluid ◽  
Chemotactic Effect ◽  
Selection Of

Chemotaxis is a spermatozoa guidance mechanism demonstrated in vitro in several mammalian species including porcine. This work focused on follicular fluid (FF), periovulatory oviductal fluid (pOF), the medium surrounding oocytes during in vitro maturation (conditioned medium; CM), progesterone (P4), and the combination of those biofluids (Σ) as chemotactic agents and modulators of spermatozoa fertility in vitro. A chemotaxis chamber was designed consisting of two independent wells, A and B, connected by a tube. The spermatozoa are deposited in well A, and the chemoattractants in well B. The concentrations of biofluids that attracted a higher proportion of spermatozoa to well B were 0.25% FF, 0.25% OF, 0.06% CM, 10 pM P4 and 0.25% of a combination of biofluids (Σ2), which attracted between 3.3 and 12.3% of spermatozoa (p < 0.05). The motility of spermatozoa recovered in well B was determined and the chemotactic potential when the sperm calcium channel CatSper was inhibited, which significantly reduced the % of spermatozoa attracted (p < 0.05). Regarding the in vitro fertility, the spermatozoa attracted by FF produced higher rates of penetration of oocytes and development of expanded blastocysts. In conclusion, porcine reproductive biofluids show an in vitro chemotactic effect on spermatozoa and modulate their fertilizing potential.

scholarly journals Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

2012 ◽  
Vol 81 (3) ◽  
pp. 229-234 ◽  
Author(s):  
Martina Lojkic ◽  
Iva Getz ◽  
Marko Samardžija ◽  
Mario Matkovic ◽  
Goran Bacic ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

Oviduct fluid extracellular vesicles regulate polyspermy during porcine in vitro fertilisation

2020 ◽  
Vol 32 (4) ◽  
pp. 409 ◽  
Author(s):  
A. S. Alcântara-Neto ◽  
M. Fernandez-Rufete ◽  
E. Corbin ◽  
G. Tsikis ◽  
R. Uzbekov ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Penetration Rate ◽  
In Vitro Fertilisation ◽  
Fertilisation Rate ◽  
Transmission Electron ◽  
Reproductive Events ◽  
Oviduct Fluid ◽  
Oviductal Fluid ◽  
Phosphate Buffered Saline

High polyspermy is one of the major limitations of porcine invitro fertilisation (IVF). The addition of oviductal fluid (OF) during IVF reduces polyspermy without decreasing the fertilisation rate. Because extracellular vesicles (EVs) have been described as important OF components, the aim of this study was to evaluate the effect of porcine oviductal EVs (poEVs) on IVF efficiency compared with porcine OF (fresh and lyophilised). OF was collected from abattoir oviducts by phosphate-buffered saline flush, and poEVs were isolated by serial ultracentrifugation. Four IVF treatments were conducted: poEVs (0.2mgmL–1), OF (10%), lyophilized and reconstituted pure OF (LOF; 1%) and IVF without supplementation (control). Penetration, monospermy and IVF efficiency were evaluated. Transmission electron microscopy showed an EVs population primarily composed of exosomes (83%; 30–150nm). Supplementation with poEVs during IVF increased monospermy compared with control (44% vs 17%) while maintaining an acceptable penetration rate (61% vs 78% respectively) in a similar way to OF and LOF. Western blotting revealed poEVs proteins involved in early reproductive events, including zona pellucida hardening. In conclusion, our finding show that poEVs are key components of porcine OF and may play roles in porcine fertilisation and polyspermy regulation, suggesting that supplementation with poEVs is a reliable strategy to decrease porcine polyspermy and improve invitro embryo production outcomes.

scholarly journals PHYTOCHEMICAL SCREENING AND ANTIAMEBIC STUDIES OF TAMARINDUS INDICA OF LEAVES EXTRACT

2019 ◽  
pp. 507-512
Author(s):  
MANSOUR ABDULNABI H MEHDI ◽  
FADEL YS ALARABI ◽  
MAZAHAR FAROOQUI ◽  
VIDYA PRADHAN
Keyword(s):  
Entamoeba Histolytica ◽  
Culture Media ◽  
Tamarindus Indica ◽  
Phytochemical Screening ◽  
Plant Leaves ◽  
High Concentration ◽  
Amebic Dysentery ◽  
In Vitro Sensitivity ◽  
Ethanolic Extracts

Objective: The present study deals with preliminary phytochemical screening of Tamarindus indica extracts and investigates its antiamebic effect against Entamoeba histolytica in vitro. Methods: E. histolytica was isolated from stools of patients with amebic dysentery and cultivated in lock-egg medium. The leaves extract was added to check its antiamebic activity. Results: The phytochemical screening shows that T. indica contains alkaloids, flavonoids, phenol, and tannins. T. indica extracts (aqueous and ethanolic) were added to culture media E. histolytica. It was observed that E. histolytica count reduced to zero after 72 h and 96 h when 15 mg/ml of aqueous and ethanolic extracts were added, respectively. It is also revealed that there is no cytotoxicity against erythrocytes even when high concentration of plant leaves extract is used. Conclusion: The in vitro sensitivity of T. indica leaves extract against the E. histolytica is established.

Effect of α-tocopherol on in vitro culture of bovine embryos

2007 ◽  
Vol 87 (4) ◽  
pp. 539-542 ◽  
Author(s):  
A. Marques ◽  
G. Antunes ◽  
P. Santos ◽  
A. Chaveiro ◽  
F. Moreira da Silva
Keyword(s):  
Culture Media ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Media Control ◽  
Oviductal Fluid ◽  
Bovine Oocytes ◽  
Key Words Antioxidant

Bovine oocytes were matured and fertilised under 5% CO2. Presumptive zygotes were co-cultured in synthetic oviductal fluid droplets, supplemented with either 0 (control), 25, 50, 100, or 200 µM of α-tocopherol. Blastocyst development rates were significantly influenced (P < 0.05) by the level of antioxidant in the culture media. Control showed a blastocyst yield of 18.46, 21.11, 27.92, and 31.66%, respectively, at α25; α50 and α100. Blastocyst yield for α200 was severely decreased, to 8.01%. An increase in overall IVF results was also observed as the concentration of α-tocopherol increased (control, 14.21%; α25, 14.35%; α50, 19.52%; α100, 21.11%), decreasing to 5.75% for the concentration of α200. The present study clearly demonstrates that α-tocopherol at a concentration of 100 µM significantly improves the proportion of oocytes that develop to the blastocyst stage. Key words: Antioxidant, α-tocopherol, reactive oxygen species, bovine, embryo

Inhibition of boar sperm hyaluronidase activity by tannic acid reduces polyspermy during in vitro fertilization of porcine oocytes

Zygote ◽  
2006 ◽  
Vol 14 (4) ◽  
pp. 275-285 ◽  
Author(s):  
Hideki Tatemoto ◽  
Isao Tokeshi ◽  
Satoshi Nakamura ◽  
Norio Muto ◽  
Tadashi Nakada
Keyword(s):  
Tannic Acid ◽  
Penetration Rate ◽  
Vitro Fertilization ◽  
Hyaluronidase Activity ◽  
High Penetration ◽  
Boar Sperm ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

SummaryThe present study was conducted to examine the effects of three polyphenols (tannic acid, apigenin and quercetin) on hyaluronidase activity and in vitro fertilization (IVF) parameters. Among them, tannic acid showed by far the strongest potency for blocking hyaluronidase activity extracted from preincubated boar sperm, causing a dose-dependent inhibition over the range of 2–10 μg/ml. When cumulus-intact and cumulus-free oocytes were inseminated in IVF medium containing tannic acid, the penetration and the polyspermy rates were significantly decreased in the presence of 10 μg/ml tannic acid compared with those in the absence of tannic acid, and the addition of 5 μg/ml tannic acid significantly reduced the polyspermy rate (p < 0.05) compared with that of the control while maintaining the high penetration rate. However, apigenin and quercetin had no effect on the rate of polyspermy. Interestingly, the incidence of polyspermy was significantly reduced in oocytes inseminated with sperm pretreated with 5 μg/ml tannic acid (p < 0.05), although the pretreatment of oocytes had no effect against the polyspermy after insemination with untreated sperm. Treatment with tannic acid caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization, nor a reduction of the proteolytic activity of acrosomal contents or the number of zona-bound spermatozoa. These data suggest that an appropriate concentration of tannic acid prevents polyspermy through the inhibition of sperm hyaluronidase activity during IVF of porcine oocytes.

scholarly journals Influence of oocyte recovery method, in vitro fertilization method and serum source on embryonic development of in vitro matured bovine oocytes

2008 ◽  
Vol 51 (3) ◽  
pp. 224-234 ◽  
Author(s):  
H. Alm ◽  
H. Torner ◽  
W. Kanitz ◽  
K. Roschlau
Keyword(s):  
Production System ◽  
Preparation Method ◽  
Blastocyst Development ◽  
Recovery Method ◽  
Sperm Preparation ◽  
Oocyte Recovery ◽  
System 2 ◽  
Blastocyst Rate ◽  
System 1

Abstract. The objective of this study was to investigate the influence of various factors on blastocyst development in a bovine in vitro embryo production system. Two production systems were originally compared: Protocol 1, which utilized oocyte recovery by slicing, 20 % fetal bovine serum with FSH (FBS+) for oocyte maturation, and sperm preparation system 1 (F1), and Protocol 2, which utilized oocyte recovery by aspiration, 5 % estrous cow serum with FSH, HCG and estradiol (ECS+) for oocyte maturation, and sperm preparation system 2 (F2). Because a significantly higher blastocyst development rate was found for Protocol 2, the different factors of oocyte recovery technique (slicing vs. aspiration), sperm preparation technique (F1 vs. F2) and serum and hormone supplementation during maturation (FBS+ vs. ECS+) were evaluated. Recovering oocytes using the alternative technique (slicing vs. aspiration) did not significantly alter blastocyst rate on Day 8 within either production system 1 (FBS+/F1) or production system 2 (ECS+/F2). Although investigations of influence of the type of fertilization method revealed no effect, a significant effect of the type of serum was observed on the blastocyst rates with ECS+ proving better as compared to the use of FBS+ (P<0.03). When embryos produced from these investigations were evaluated by differential staining, the number of ICM cells did not differ among treatment, but the number of TE cells, and thus the total cell number, and the ICM : TCN ratio, was significantly increased when oocytes were matured in ECS+ supplemented medium, regardless of sperm preparation method. In conclusion, serum supplementation, but not oocyte recovery method or sperm preparation method, was responsible for the difference in blastocyst rate between the two original IVP protocols.

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