198 FUNCTIONAL EVIDENCE FOR THE EXISTENCE OF AN OVIDUCTAL FACTOR THAT INDUCES ZONA PELLUCIDA HARDENING AND REGULATES POLYSPERMY IN THE PIG AND COW

2008 ◽  
Vol 20 (1) ◽  
pp. 178 ◽  
Author(s):  
S. Cánovas ◽  
L. Grullón ◽  
R. Romar ◽  
C. Matás ◽  
M. Avilés ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Control Groups ◽  
Digestion Time ◽  
Vitro Fertilization ◽  
The Mean ◽  
Oviductal Fluid ◽  
Bovine Oocytes

Many differences between in vivo and in vitro fertilization (IVF) efficiency in mammals are related to the differences between IVF media and oviductal fluid. One of the best known examples is the frequency of polyspermy observed under in vitro conditions in cattle (Roh S et al. 2002 J. Vet. Med. Sci. 64, 667–671) and, in particular, in pigs (Coy P and Romar R 2002 Reprod. Fertil. Dev. 14, 275–286). Zona pellucida (ZP) resistance to pronase digestion (ZP hardening) has been considered as a postfertilization event contributing to the block of polyspermy in mammals (Green D 1997 Rev. Reprod. 2, 147–156). However, pig and cow unfertilized ovulated oocytes show a ZP hardening of hours or days (Katska L et al. 1999 Reprod. Dom. Anim. 34, 255–259; Kolbe T and Holtz W 2005 Theriogenology 63, 1695–1705) compared with the minutes or seconds observed in the in vitro-matured oocytes, even after fertilization (Coy P et al. 2002 Reproduction 124, 279–288; Coy P et al. 2005 Reproduction 129, 19–26). Consequently, we propose the existence of an oviductal factor that induces ZP hardening before any contact of the oocyte with the sperm, thus regulating polyspermy. Porcine and bovine oviductal fluid was obtained by aspiration of oviducts collected at the slaughterhouse and stored frozen. In vitro-matured porcine and bovine oocytes were incubated for 30 min in the oviductal fluid, washed thoroughly in fresh medium, and either assessed for ZP digestion time or in vitro fertilized. The results, analyzed by ANOVA, showed a very strong ZP hardening in oviductal-treated oocytes (2866.83 � 94.4 s in the pig and 4301.1 � 441.7 s in the cow) compared with control oocytes (63.5 � 2.9 s in the pig and 124.2 � 5.9 s in the cow). Moreover, the percentage of monospermy for the oviductal-treated oocytes was significantly higher in both species (50.0 � 10.0% in the pig and 91.7 � 3.0% in the cow) compared with the control groups (5.56 � 3.8% in the pig and 80.8 � 3.5% in the cow). Percentage penetration did not change in porcine oocytes but decreased in bovine oocytes (58.1 � 3.3 v. 38.4 � 3.3, P ≤ 0.001), whereas the mean number of sperm per oocyte decreased for the porcine-treated oocytes (2.7 � 0.2 v. 8.2 � 0.4, P ≤ 0.001) and did not change for the bovine oocytes. These results support the hypothesis that an oviductal factor induces ZP hardening, contributing to the control of polyspermy in the pig and cow, and that it could be used to improve the output of IVF. Supported by MEC and FEDER (AGL2006-03495).

In vitro fertilization and cleavage of in vivo matured bovine oocytes

1984 ◽  
Vol 22 (2) ◽  
pp. 151-165 ◽  
Author(s):  
T. Greve ◽  
D. Bousquet ◽  
W.A. King ◽  
K.J. Betteridge
Keyword(s):  
In Vitro Fertilization ◽  
Vitro Fertilization ◽  
Bovine Oocytes

173 COMPARISON OF FERTILIZATION RATES OF EQUINE OOCYTES USING IN VITRO FERTILIZATION, PERIVITELLINE AND INTRACYTOPLASMIC SPERM INJECTIONS

2012 ◽  
Vol 24 (1) ◽  
pp. 198
Author(s):  
D. R. Sessions ◽  
J. K. Graham ◽  
E. M. Carnevale
Keyword(s):  
In Vitro Fertilization ◽  
Normal Embryo ◽  
Fertilization In Vitro ◽  
Perivitelline Space ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Chi Square Analysis ◽  
Bovine Oocytes

In vitro fertilization in horses has had limited and inconsistent success for oocytes matured in vivo or in vitro. However, oocytes transferred to the oviducts of inseminated recipient mares are consistently fertilized and result in normal embryo development. Potentially, equine sperm are unable to penetrate the zona pellucida (ZP) in vitro, which could be a result of failure of capacitation or hyperactivation. We hypothesised that ZP of equine oocytes impairs fertilization in vitro and bypassing the ZP would be beneficial for fertilization rates. Oocytes were retrieved using transvaginal follicle aspirations from oestrous mares 22 to 24 h after administration of a gonadotropin-releasing hormone analog (deslorelin, 1.1 mg). After culture for 20 h in TCM-199 with Earle's salts and 10% FCS, oocytes were assigned to 1 of 3 groups: 1) IVF, equine (n = 9) and bovine (n = 80) cumulus–oocyte complexes co-incubated with sperm; 2) PVI, sperm injections into the perivitelline space of equine (n = 13) and bovine (n = 11) oocytes; and 3) ICSI, intracytoplasmic sperm injections of equine oocytes (n = 13). Fresh sperm from 2 stallions was used. For IVF, sperm were treated with 20 μM dilauroyl phosphatidylcholine (PC12) to induce capacitation but not the acrosome reaction; sperm for PVI and ICSI were treated with 40 μM PC12 to capacitate and acrosome-reacted sperm. For IVF, each equine oocyte was co-incubated with 8 to 11 bovine oocytes and 250 000 sperm in fertilization media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607) for 18 h. For PVI, 10 to 15 sperm were injected into the perivitelline space of each equine and bovine oocyte and for ICSI, 1 sperm was injected into the cytoplasm of each equine oocyte. Oocytes were then placed in embryo culture medium (DMEM-F12 and 10% FCS) for 24 h before examination for cleavage. Cleavage rates for each group were compared using chi-square analysis. After IVF or PVI, no equine oocytes cleaved; in comparison, 11 of 13 (85%) cleaved in the ICSI group. However, of the bovine oocytes, 10 of 80 (12%) cleaved after IVF and 1 of 11 (9%) cleaved after PVI with equine sperm. An additional 6 PVI injections with equine (n = 6) oocytes and sperm were stained with Hoechst 33324 to determine sperm localization within the oocytes. All injected sperm remained in the perivitelline space and did not appear to fuse with the oolemma or enter the ooplasm. The results of this study suggest that the equine sperm prepared with PC12 were unable to penetrate the oolemma. In our experiment, sperm treated with PC12 resulted in high fertilization rates when placed directly into the ooplasm during ICSI; however, the sperm were unable to penetrate the ZP or oolemma. Additional research is needed to determine the cause of penetration failure and whether this is due to inadequate sperm treatments or a missing oviducal component in our in vitro systems. Funding for this project was provided by the benefactors for the Preservation of Equine Genetics and the Cecil and Irene Hylton Foundation.

In vitro fertilization of bovine oocytes matured in vivo and collected at laparoscopy

1986 ◽  
Vol 25 (1) ◽  
pp. 117-133 ◽  
Author(s):  
R.D. Lambert ◽  
M.a. Sirard ◽  
C. Bernard ◽  
R. Béland ◽  
J.E. Rioux ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Vitro Fertilization ◽  
Bovine Oocytes

Influence of hardening of the zona pellucida on in vitro fertilization of bovine oocytes

1989 ◽  
Vol 32 (5) ◽  
pp. 767-777 ◽  
Author(s):  
L. Katska ◽  
P. Kauffold ◽  
Z. Smorag ◽  
U. Duschinski ◽  
H. Torner ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Vitro Fertilization ◽  
Bovine Oocytes

204 AMINO-REACTIVE CROSSLINKER BIS(SULFOSUCCINIMIDYL)SUBERATE INDUCES ZONA PELLUCIDA HARDENING AND REDUCES PENETRATION IN PIG IN VITRO FERTILIZATION

2008 ◽  
Vol 20 (1) ◽  
pp. 181
Author(s):  
L. Grullón ◽  
S. Cánovas ◽  
C. Matás ◽  
R. Romar ◽  
P. Coy
Keyword(s):  
Zona Pellucida ◽  
Control Group ◽  
Digestion Time ◽  
Vitro Fertilization ◽  
Amide Bonds ◽  
High Penetration ◽  
The Mean ◽  
Pronuclear Formation ◽  
Dose Dependent Increase

Zona pellucida (ZP) hardening is considered to be the final step in the prevention of polyspermy during fertilization in mammals. However, unfertilized pig oviductal oocytes show a resistance of hours or days to pronase digestion (Broermann et al. 1989 J. Anim. Sci. 67, 1324–1329). We previously demonstrated that the amino-reactive crosslinker DSP is effective in inducing ZP hardening and improves the monospermy levels at pig IVF (Coy et al. 2007, Reprod. Dom. Anim., in press). In this study, a different chemical crosslinker, BS3 [bis(sulfosuccinimidyl) suberate], which also forms stable amide bonds among proteins, was used to evaluate its effect on ZP digestion time, penetration, male pronuclear formation and monospermy percentages, and the mean number of sperm per oocyte. In experiment 1, porcine in vitro-matured oocytes (n = 300) were incubated for 30 min at 0, 0.06, 0.30, or 0.60 mg mL–1 of BS3 in TALP medium and assessed for ZP digestion time in 0.5% pronase solution. The results (analyzed by ANOVA in all the experiments) showed a significant (P ≤ 0.01) dose-dependent increase in ZP hardening, from 69.0 s in the control to 426.3, 2028.3, and 2979.2 s, respectively, for the different BS3 concentrations. In experiment 2, oocytes (n = 473) were fertilized in vitro after no treatment or treatment with BS3 at 0.06, 0.30, and 0.60 mg mL–1. Fresh ejaculated spermatozoa were selected by Percoll� gradient 45:90. Oocytes were inseminated with 105 sperm mL–1, which resulted in high penetration and polyspermy percentages in the control group (83.1 and 89.9%, respectively). However, for the BS3-treated oocytes, significant differences compared with the control group (P ≤ 0.001) were observed in all 3 groups, showing penetration percentages of 22.2, 18.1, and 21.5%, respectively, and monospermy percentages of 100, 88.2, and 95.0%, respectively. The mean numbers of sperm per oocyte were 1.0, 1.1, and 1.05 for the BS3 groups, which were significantly different from 5.0 for the control group. In conclusion, BS3 can be used to induce ZP hardening in the pig and regulate polyspermy in IVF systems, although additional experiments are necessary to find the optimal concentration to improve the penetration percentages with high levels of monospermy. Granted by MEC and FEDER (AGL2006-03495).

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

Growth and Development of Children Conceived by In Vitro Fertilization

PEDIATRICS ◽  
1992 ◽  
Vol 90 (3) ◽  
pp. 424-429
Author(s):  
Joseph M. Brandes ◽  
Joseph Itzkovits ◽  
Anat Scher ◽  
Miriam Sarid ◽  
Israel Thaler ◽  
...  
Keyword(s):  
Birth Weight ◽  
In Vitro Fertilization ◽  
Gestational Age ◽  
Head Circumference ◽  
Healthy Population ◽  
Control Groups ◽  
Vitro Fertilization ◽  
General Physical ◽  
And Control

To assess the physical and mental development of infants born after in vitro fertilization (IVF), we performed a general physical and developmental examination (Bayley and Stanford-Binet scales) on a cohort of 116 IVF children, conceived and born at our institution between February 1985 and March 1989, and on 116 non-IVF matched controls. Study and control groups were each composed of 66 singletons, 19 pairs of twins and 4 sets of triplets, whose age at examination ranged from 12 to 45 months. The developmental indices of IVF infants were within the normal range and did not differ from those of their matched controls. The indices were positively correlated to gestational age, birth weight, head circumference at birth and at examination, and mother's education. Mean birth weight, gestational age, and birth weight percentile of IVF infants were lower than the mean of the healthy population. Mean percentiles of weight and length at examination (mean age 22.4 months) were equally low but did not differ from those of the matched controls. However, mean percentiles of head circumference at birth and at examination compare well with the normal mean, both in IVF and control groups. Twins and triplets (IVF and controls) had significantly lower physical and mental indices as compared to singletons.

Sign in / Sign up

Export Citation Format

Share Document