203 PRETREATMENT OF BOVINE SPERM OR OOCYTE WITH ANTIBODY TO LIPOCALIN-TYPE PROSTAGLANDIN D SYNTHASE INHIBITS FERTILIZATION

2008 ◽  
Vol 20 (1) ◽  
pp. 180
Author(s):  
R. F. Gonçalves ◽  
V. H. Barnabe ◽  
G. J. Killian
Keyword(s):  
Polyclonal Antibody ◽  
Gradient Centrifugation ◽  
Cumulus Cells ◽  
Fertilization In Vitro ◽  
Prostaglandin D Synthase ◽  
Rabbit Polyclonal Antibody ◽  
Development In Vitro ◽  
Triton X ◽  
Bovine Oocytes

Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (UTF), and as fertility-associated protein in the Holstein bull seminal plasma, but its function is unclear. A previous study demonstrated that L-PGDS is associated with the bovine zona pellucida, and that antibody incubated with UTF decreased embryo development in vitro. This study was conducted to determine whether IVF of bovine oocytes would be affected by pretreating either the sperm or oocytes, or both, with L-PGDS antibody. In vitro-matured bovine oocytes were incubated for 1 h in IVF TALP medium supplemented with penicillamine, hypotaurine, epinephrine, and heparin containing (a) no antibody, or (b) a rabbit polyclonal antibody against recombinant bovine L-PGDS (α L-PGDS; 1:2000). Frozen–thawed spermatozoa were washed by a 45:90% layered Percoll gradient centrifugation and incubated for 1 h in IVF TALP with (a) no antibody, or (b) α L-PGDS. For this study we had 4 different treatments: (1) no antibody (control), (2) α L-PGDS at fertilization time, (3) α L-PGDS-treated oocytes, or (4) α L-PGDS-treated sperm. Oocytes were inseminated with 10 � 104 washed spermatozoa in 4-well culture dishes. After 18 h (39�C, 5% CO2 in air), oocytes were vortexed to remove cumulus cells and accessory spermatozoa, and fixed in 3.7% paraformaldehyde and 10% Triton X–100 for 1 h. Oocytes were washed and transferred to a solution with PBS, 0.3% BSA, and 1% Triton for 1 h, stained with Hoechst 33342, and observed in the presence of 2 pronuclei in the cytoplasm. There were 4 replicates of 200 to 250 oocytes for fertilization assays. Data were analyzed by SAS. Addition of α L-PGDS with sperm, oocytes, or both significantly decreased fertilization (P < 0.05) compared with the control: (1) 89.2 � 2.0%; (2) 19.4 � 2.0%; (3) 27.2 � 3.1%; or (4) 14.1 � 3.4%. These studies demonstrated that a rabbit polyclonal antibody against recombinant bovine L-PGDS reacts with both oocytes and spermatozoa, resulting in inhibition of fertilization in vitro, and has a possible role in bovine fertilization. This study was supported by FAPESP #2007/00363-5 and #2006/06008-0, Brazil.

239 INFLUENCE OF BREED PREDOMINANCE ON MATURATION COMPETENCE, FERTILIZATION, AND DEVELOPMENT IN VITRO OF BOVINE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
J. Pelaez ◽  
H. Hernandez-Fonseca ◽  
A. Pirela ◽  
F. Baez ◽  
P. Villamediana ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Cleavage Rate ◽  
Ethanol Acetic Acid ◽  
Chi Squared ◽  
Development In Vitro ◽  
Bovine Oocytes

The purpose of this research was to compare the competence of bovine oocytes of different breed predominance (Bos taurus v. Bos indicus) to mature and to be fertilized. This was done through the collection, selection, maturation, and fertilization of oocytes from slaughtered cows, predominantly either B. taurus or B. indicus. Only cows that were at least 5/8 B. taurus or 5/8 B. indicus, according to a series of phenotypic characteristics, such as the presence of a hump, dewlap, length of the ears, and others, were selected. To obtain cumulus–oocyte complexes, ovarian follicles (3 to 10 mm in diameter) were aspirated, and only oocytes with 2 or more layers of cumulus cells, an intact zona pellucida, and a homogeneous granular cytoplasm were selected. After selection, oocyte maturation [in vitro maturation (IVM)] and IVF were done. Frozen–thawed semen was used from one Brahman bull (B. indicus). For the evaluation of IVM as for IVF, oocytes were fixed for approximately 24 h at 4°C in a solution of ethanol : acetic acid (3 : 1). They were then stained with 1% acetic orcein. A chi-squared test was performed for all reported rates. The rate of maturation of oocytes from cows with a predominant B. indicus phenotype was 66.93%, whereas cows with a B. taurus phenotype reached 43.10% (P < 0.001). Regarding the fertilization rate, predominantly B. indicus females had 43.68% of oocytes normally fertilized and 41.74% of oocytes were abnormally penetrated. This category included polyspermic and asynchronic (abnormally developed pronucleus) oocytes. In cows with B. taurus predominance, 31.96% of oocytes were normally penetrated and 46.39% were abnormally penetrated by spermatozoa (no significant differences were found). The rate of non-fertilized oocytes was significantly different (P < 0.05) among B. indicus and B. taurus oocytes (6.79 and 17.52%, respectively). A small and nonsignificant proportion of degenerated oocytes resulted in both groups (7.79% for B. indicus and 4.14% for B. taurus). The cleavage rate was not different among phenotypic groups (36.12 and 32.30%, respectively, for B. indicus and B. taurus). In conclusion, the present results indicate that oocytes from predominantly B. indicus cows were more competent than oocytes from cows with a predominance of the B. taurus breed. Nonetheless, this superiority was not evident in terms of cleavage rates. Semen from other B. indicus and B. taurus breeds must be tested to clarify any breed interactions.

Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro

Reproduction ◽  
2000 ◽  
pp. 127-135 ◽  
Author(s):  
W Bone ◽  
NG Jones ◽  
G Kamp ◽  
CH Yeung ◽  
TG Cooper
Keyword(s):  
Zona Pellucida ◽  
Glucose Utilization ◽  
Cumulus Cells ◽  
Glycolytic Enzyme ◽  
Male Rats ◽  
Fertilization In Vitro ◽  
Swimming Pattern ◽  
Principal Piece ◽  
Free Media

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Shiori Ashibe ◽  
Kanade Irisawa ◽  
Ken Yokawa ◽  
Yoshikazu Nagao
Keyword(s):  
Treatment Group ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Free Calcium ◽  
Parthenogenetic Development ◽  
Early Embryos ◽  
Bovine Oocytes

Summary Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

Fertilization in vitro of bovine oocytes after various sperm procedures

1983 ◽  
Vol 20 (6) ◽  
pp. 651-660 ◽  
Author(s):  
Y. Fukui ◽  
M. Fukushima ◽  
H. Ono
Keyword(s):  
Fertilization In Vitro ◽  
Bovine Oocytes

381 PRODUCTION OF A TRANSGENIC PIGLET BY A NEW SPERM INJECTION TECHNIQUE

2006 ◽  
Vol 18 (2) ◽  
pp. 297
Author(s):  
H. Y. Yong ◽  
C. Murphy ◽  
A. Rieke ◽  
L. Lai ◽  
Y. Hao ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Fluorescent Protein ◽  
Injection Technique ◽  
Motile Sperm ◽  
Gfp Expression ◽  
Injection Process ◽  
Development In Vitro ◽  
Green Fluorescent ◽  
Triton X

The technique for intracytoplasmic sperm injection (ICSI) has, until now, focused on scoring the tail of the sperm prior to catching and aspiration into the injection pipette. This is in spite of the fact that damage to the head would more closely simulate what occurs during normal fertilization. In addition, to aid in visualizing the injection process so that a reduced volume can be injected, the oocyte is generally centrifuged to clear a portion of the cytoplasm. Thus, with conventional ICSI, the sperm are immobilized with polyvinylpyrrolidone, repeatedly frozen and thawed, treated with DTT or Triton X-100, and severed between the head and tail; the oocyte is centrifuged or activated. All of the above treatments are designed to compensate for the intrinsic defects in conventional ICSI. Our objective was to use a modified ICSI procedure whereby aggressively motile sperm were captured onto the broken tip of an injection pipette and then injected into noncentrifuged oocytes. Damage to the head of the sperm occurred on the pipette or while pushed through the zona pellucida. These procedures are based on the work of Yong et al. 2003 Hum. Reprod. 18, 2390, where they achieved an improvement in development in vitro as compared to conventional methods. Ovaries were collected from prepubertal gilts, and oocytes were aspirated and matured in vitro. Sperm were collected from a transgenic boar carrying the green fluorescent protein (GFP) and frozen. After thawing, aggressively motile sperm were captured and injected through the zona pellucida and into the cytoplasm of the in vitro-matured oocytes. A total of 452 injected oocytes (43-171 oocytes per recipient) were surgically transferred into the oviduct of six surrogate gilts. Two gilts (33%) became pregnant. One gave birth to a healthy male piglet. GFP expression was observed in the nose and hooves by direct epifluorescent examination of the newborn piglet. This pattern of GFP expression is identical to that in non-ICSI-derived GFP pigs in this line. This result showed for the first time that this new sperm injection technique could be used for production of a viable transgenic piglet using in vitro-matured oocytes and frozen-thawed sperm.

346 BENEFICIAL EFFECTS OF ACETYL-l-CARNITINE TREATMENT DURING IVM ON POST-FERTILIZATION DEVELOPMENT OF BOVINE OOCYTES IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 280 ◽  
Author(s):  
T. Yamada ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
Energy Production ◽  
Metabolic Regulation ◽  
Cellular Proliferation ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Acid Oxidation ◽  
Oocyte Cytoplasm ◽  
Peripheral Type ◽  
Bovine Oocytes

The lower competence of in vitro-matured oocytes for post-fertilization development is attributed to the lack of physiological factors in in vitro maturation (IVM) that regulate maturation events which occur exclusively in the cytoplasm of oocytes. It has been found recently that mitochondrial function plays an important role in regulation of oocyte developmental competence via metabolic regulation of energy production. Acetyl-l-carnitine (ALC) is known to enhance fatty acid oxidation and energy production in the mitochondria, and to exert enhancing effects on cellular proliferation and survival. In this experiment, we examined the effects of ALC on IVM and post-fertilization development of bovine oocytes. Cumulus-oocyte complexs (COCs) were aspirated from 2-5 mm follicles of ovaries from a slaughterhouse. COCs were cultured in IVM medium (mSOFaa+estradiol+hCG+BSA) with or without ALC (10 mM) for 24 h at 39�C under 5% CO2 in air, and then fertilized according to the conventional method. After 6 h of insemination, presumptive zygotes were freed from cumulus cells by repeated pipetting and cultured in mSOFaa with 1% FCS at 39�C under 5% CO2, 5% O2, and 90% N2. At 48 h post-fertilization, the rates of cleaved embryos were assessed. The cleaved embryos were transferred to mSOFaa with 5% FCS and cultured for additional 6 days at 39�C under 5% CO2, 5% O2, and 90% N2. The percentages of embryos developing to the blastcyst stage were assessed on Days 6, 7, and 8 (fertilization = Day 0), and the data were analyzed for statistically significant differences with the t-test. For examinination of mitochondrial organization in oocytes at different maturation stages, oocytes were stained for active mitochondria with MitoRed (1 �M in IVM medium for 2 h at 37�). When COCs were matured in medium without (control) or with ALC, although the rates of post-fertilization cleavage of oocytes were 60% to 70% despite the presence or absence of ALC, ALC significantly (P < 0.05) increased the rates of cleaved embryos forming blastcysts on Days 6, 7, and 8 (30%, 36%, 40%) compared with those in the control (13%, 21%, 34%). We next examined effects of ALC treatment during IVM on active mitochondria distribution in oocytes. In 75% of immature oocytes, active mitochondria localized in the periphery of the oocytes (peripheral type). After 24 h of IVM without ALC, while 17% of oocytes remained in a peripheral type, 44% showed some migration of active mitochondria toward the center of the oocytes (semiperipheral type) and 39% presented a diffused distribution of active mitochondria in the whole oocyte cytoplasm (diffused type). On the other hand, in ALC treated oocytes, 60% of the oocytes presented a diffused type, 25% exhibited a semiperipheral type, and 15% had still maintained a peripheral distribution. These results provide the first evidence that ALC treatment during IVM of bovine oocytes enhances their post-fertilization development to the blastcyst stage and enhances the frequency of oocytes that exhibit an extensive relocation (diffused type) of active mitochondria to the inner oocyte cytoplasm.

6 TIMING OF DEADENYLATION OF GDF-9 AND CYCLIN 3′ UTR CONSTRUCTS IN BOVINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 121
Author(s):  
D. J. Walker ◽  
C. J. Wilusz ◽  
G. E. Seidel Jr
Keyword(s):  
In Vitro Maturation ◽  
Control Point ◽  
Tail Length ◽  
Cumulus Cells ◽  
Cyclin B1 ◽  
Defined Medium ◽  
Untranslated Regions ◽  
Labeled Rna ◽  
Bovine Oocytes

The maternal pool of mRNA undergoes major changes during oocyte maturation and early embryonic development. Specific genes are activated or degraded in response to changes in poly-(A) tail length. However, little is known about how the oocyte targets specific transcripts for degradation or translation in a timely manner. The objective of this study was to determine how poly-(A) tail length of different transcripts is affected in bovine oocytes by time of in vitro maturation. Cyclin B1 and GDF-9 32 untranslated regions (UTRs) were cloned into modified p-GEM plasmids containing a poly-(A) tract of 60 or 0 adenosines (A60 or A0, respectively). Each 32 UTR was transcribed in vitro with (A60) or without (A0) a poly-(A) tail to generate UTP32-labeled RNA. Transcriptions producing at least 200 000 counts per min (cpm) per �L were used for subsequent injections into denuded bovine oocytes. Cumulus-oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries (n = 216) were vortexed to remove cumulus cells immediately after aspiration, after 3 h of in vitro maturation, or after 19 h of maturation in a chemically defined medium supplemented with FSH, LH, EGF, and cysteamine. After vortexing, denuded oocytes were injected and snap frozen, or matured in vitro for 1 or 3 h. Eight oocytes were injected with ~0.5 nL (~100 cpm/oocyte) labeled RNA at each time point in 3 replicates. Total RNA was isolated from injected oocyte pools and loaded onto a 5% denaturing acrylamide gel for size separation. Radiolabeled A0 was used as a control point of reference for deadenylation. Gels were dried, and RNA was visualized on a phosphoimager after 24 h exposure to a phosphor screen. Changes in polyadenylation status (transcript size) were evaluated by comparing shifts in bands from gene-specific A60

199 EFFECT OF HEPARIN CONCENTRATION ON BOVINE PREIMPLANTATIONAL DEVELOPMENT IN VITRO USING SEX-SORTED SPERM

2008 ◽  
Vol 20 (1) ◽  
pp. 178
Author(s):  
S. A. Chaubal ◽  
T. L. Nedambale ◽  
J. Xu ◽  
C. Shaffer ◽  
T. Kilmer ◽  
...  
Keyword(s):  
Embryo Development ◽  
Research Education ◽  
Heparin Concentration ◽  
State Research ◽  
Specific Manner ◽  
Sorting Process ◽  
Recorded Data ◽  
Development In Vitro ◽  
Bovine Oocytes

The objective of this study was to examine the effect of heparin on bovine IVF and to improve the efficiency of IVF production by using sex-sorted sperm. The fertility performance of sex-sorted and unsorted semen from 4 bulls was compared to determine the optimal heparin concentration during preimplantational embryo development. A total of 7615 matured bovine oocytes were randomly allocated among different heparin concentrations (0, 2.5, 5, 10, 20, 40, 60, 80, and 100 μg mL–1) in Brackett-Oliphant medium and coincubated with either sex-sorted or unsorted sperm for 6 h. Presumptive zygotes were cultured in CR1aa+ 6 mg mL–1 of BSA in 5% O2 , 5% CO2 and 90% N2 at 39°C until Day 8 (Day 0, culture post-IVF). Cleavage rates at Day 2 and embryo development to blastocyst (BL) at Day 8 were recorded. Data (4 replicates) were analyzed by a general linear model (SPSS 11.0, SPSS Inc., Chicago, IL). The optimal heparin concentration for each treatment was determined as the lowest value from those groups that resulted in the highest BL rates. The results (Table 1) demonstrated that a differential requirement of heparin concentration was important for the highest preimplantational BL development between sexed sperm and unsorted control within each bull. By optimizing heparin concentration, in 3 out of 4 (75%) bulls, the in vitro BL development with sex-sorted sperm could be increased to a level that was comparable to the highest BL rate from unsorted sperm (bulls A, B, and C, P > 0.05). A higher heparin concentration was required for optimal BL development in bulls A and C; however, a lower concentration was desirable for bulls B and D, indicating that a partial capacitation to the sperm may have taken place in bulls B and D during the sorting process, as reported by Lu and Seidel (2004 Theriogenology 62, 819–830). The fertility of sorted sperm from bull D (1 out of 4, 25%) was adversely affected, even after heparin optimization for BL development (P < 0.05). This result suggests that sperm sorting could affect the IVF fertility of sorted sperm in a bull-specific manner, but it was not significant for all bulls. Table 1. Blastocyst (BL) development in bovine IVF after heparin optimization using sorted and unsorted sperm This project was supported by the SBIR program under a USDA Cooperative State Research, Education, and Extension Service (CSREES) grant to F. Du (USDA #2006-03069).

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