Efficiency Of In Vitro Fertilization Research Articles

Culture of viable human blastocysts in defined sequential serum-free media.

In human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development, resulting in a 10-15% implantation rate. However, in other mammalian species, the transfer of cleavage stage embryos, which normally reside in the oviduct, to the uterus results in a significantly lower implantation rate compared with blastocysts. It is therefore proposed that, in order to increase implantation rates in human IVF, one has to move to extended culture and transfer at the blastocyst stage. The transfer of blastocysts will not only help synchronize the embryo with the female tract but will facilitate the identification of those embryos with little or no developmental potential. In order to culture viable blastocysts it is important to use more than one culture medium to cater for the changing requirements of the preimplantation embryo as it develops and differentiates. If sequential culture media are not used, one can obtain blastocysts but their resultant viability is low. The use of sequential serum-free media in human IVF has resulted in > 50% of embryos becoming blastocysts with an implantation rate of approximately 50%. Further advances in human embryo culture should come from the replacement of protein with the glycosaminoglycan hyaluronate, which is more suitable than albumin in supporting implantation in the mouse, and which will eliminate biological variation and possible contamination from blood products. With the routine culture of human blastocysts will come the introduction of non-invasive tests of embryo viability, capable of identifying those blastocysts most likely to develop from a given cohort. As the implantation rate of blastocysts is higher than that of the cleavage stage embryo, fewer embryos will be required for transfer in order to establish a successful pregnancy, thereby reducing the number of multiple gestations and increasing the overall efficiency of human IVF.

Progression of meiosis in porcine oocytes matured in vitro and the polyspermy problem

The high incidence of polyspermy is still an unresolved problem for the production of in vitro-produced porcine embryos. In this work, we modified the usual sperm processing sequence for in vitro fertilization (IVF), and the spermatozoa from four boars were frozen directly at a low sperm concentration of 20 × 106 sperm/mL (high pre-freezing sperm dilution group; F20), thawed and processed for IVF in three replicates. Spermatozoa from the same boars frozen at a conventional concentration (1000 × 106 sperm/mL) were used as the control group. The post-thaw sperm quality evaluation demonstrated that despite there being no differences in the percentage of motile spermatozoa between groups, the proportion of live spermatozoa with intact acrosomes was significantly higher in the F20 group than in the control. The in vitro penetration rate was also similar between groups; however, the co-incubation of oocytes with F20 sperm increased monospermy, IVF efficiency, cleavage rate and the efficiency of blastocyst formation compared with the results for oocytes co-incubated with control spermatozoa. These results indicate, for the first time, that a high pre-freezing sperm dilution increases monospermy without affecting penetration rates, thereby increasing blastocyst formation.

The production of ruminant embryos in vitro

The potential for dramatic increases in genetic gain, and the production of transgenic animals with highly desired production, and disease resistance characteristics through DNA transfection techniques, in pronuclear oocytes and embryonic stem cells, and nuclear transplantation for embryo multiplication and transfer (EMT) has resulted in increased research or the production of embryos in vitro. The primary techniques which are essential for embryo production are maturation of oocytes in vitro (IVM), in vitro fertilization (IVF) and embryo culture in vitro. Combining these techniques enables the large scale production of mature and fertilized oocytes for the introduction of genes, embryos for the production of embryonic stem cells and embryos for transfer, and oocytes and embryos for EMT. There is considerable margins available to improve the efficiency of IVM, IVF and embryo culture techniques. While the majority of oocytes will undergo nuclear maturation by culture in medium containing LH, FSH and oestrogen or oestrus serum, their complete nuclear maturation and viability need to be improved. There are problems for achieving fertilization in vitro for many desirable sires and this needs to be solved by determining universal sperm capacitation techniques. Embryo culture is still apparently sub-optimal although some reports indicate that in vivo fertilized embryos can be cultured without a dramatic loss of viability. It is possible to produce dramatic improvements in the production capacity for ruminant embryos by relatively simple adjustments to technique but further improvements may require more basic research to ascertain the cellular physiology of gamete maturation, fertilization and early embryonic development.

Correlation of sperm viability with gamete interaction and fertilization in vitro in the cheetah (Acinonyx jubatus).

Sperm-oocyte interaction in vitro was studied in the cheetah, a species known to produce poor quality ejaculates and to experience low rates of fertility. Twelve female cheetahs were injected (i.m.) with eCG followed by hCG 84 h later. Twenty-four to 26 h post hCG, each was subjected to laparoscopic oocyte aspiration. A sperm motility index (SMI) was calculated for each of 9 cheetah sperm donors that produced ejaculates averaging 41.3 +/- 22.9 x 10(6) motile sperm and 28.4 +/- 4.9% structurally normal sperm. Each ejaculate was used to inseminate cheetah oocytes from 1 or 2 females and salt-stored, domestic cat oocytes. The presence of ovarian follicles (greater than or equal to 1.5 mm in diameter) showed that all females responded to exogenous gonadotropins (range, 11-35 follicles/female). A total of 277 cheetah oocytes was collected from 292 follicles (94.9% recovery; 23.1 +/- 2.2 oocytes/female). Of these, 250 (90.3%) qualified as mature and 27 (9.7%) as degenerate. Of the 214 mature oocytes inseminated, 56 (26.2%) were fertilized, and 37 (17.3%) cleaved to the 2-cell stage in vitro; but the incidence of in vitro fertilization (IVF) varied from 0 to 73.3% (p less than 0.001) among individual males. When oocytes from individual cheetahs (n = 5) were separated into two groups and inseminated with sperm from a male with an SMI greater than 0 after 6 h coincubation versus an SMI = 0 at this time, the mean fertilization rates were 28/44 (63.6%) and 0/37 (0%), respectively (p less than 0.05). Of the 117 domestic cat oocytes coincubated with cheetah sperm, 96.6% contained 1 or more cheetah sperm in the outer half of the zona pellucida (ZP). Although the mean number of cheetah sperm penetrating the outer ZP of the cat oocyte was similar (p greater than 0.05) among all males, there was a positive correlation between the number of sperm reaching the inner half of the ZP and fertilization rate in vitro (r = 0.82; p less than 0.05). Compared to IVF efficiency in the domestic cat and tiger as reported in earlier studies, IVF efficiency in the cheetah is low. Because oocytes from 11 of 12 cheetahs were fertilized in vitro, there is no evidence that the female gamete is incompetent. Although sperm pleiomorphisms may contribute to poor reproductive performance, examination of the data on the basis of individual sperm donors reveals that effective gamete interaction in the cheetah is dictated, in part, by sperm motility.