Maturation and fertilization of porcine oocytes in vitro

Four experiments were conducted to study 1) factors affecting porcine oocyte maturation in culture medium and 2) a new method for oocyte maturation outside the porcine body. In Experiment 1, five groups of oocytes were cultured in m-TCM199 or m-KRB medium for 24 to 28, 32 to 36 or 40 to 42 hours and then were fertilized in vitro. The cleavage rate (two to four-cell stage) of oocytes cultured for 32 to 36 hours was significantly higher than those of the other oocytes. The results indicate that a suitable culture period for the in vitro maturation of porcine oocytes is 32 to 36 hours. In Experiment 2, four groups of oocytes were cultured in m-KRB or m-KRB supplemented with PFF, PMSG or FSH for in vitro maturation, and the cleavage rates of oocytes were 7.94, 22.56, 30.23 and 23.26%, respectively, after in vitro fertilization. The results show that porcine follicular fluid (PFF) and gonadotrophins added to the culture medium promote porcine oocyte maturation in vitro. In Experiment 3, oocytes were cultured in m-KRB or m-TCM199, supplemented with both gonadotrophin and pocine folliclar fluid for maturation in vitro. After fertilization in vitro, the cleavage rates of oocytes were 26.32 and 27.93% for the two media. The results indicate that the difference between m-KRB and m-TCM199 was insignificant when the media were used to culture porcine oocytes. But there was a significant difference when PFF and gonadotrophins were added to the basic media. In Experiment 4, porcine oocytes were transferred into the reproductive tracts of other animals for maturation. After 34 to 36 hours, the oocytes were collected and fertilized in vitro. The cleavage rates of oocytes were 10.42, 28.45, 3.33 and 36.36%, respectively, for the oocytes matured in mouse uterine horns, rat uterine horns, rat oviducts or rabbit oviducts. The results show that porcine oocytes can be matured in the reproductive tracts of other animals.     

Maturation, fertilization and complete development of porcine oocytes matured under different systems

This study was designed 1) to determine the effectiveness of 2 in vitro maturation systems commonly employed to produce nuclear and cytoplasmically mature pig oocytes, 2) to assess the effects of boar, sperm concentration and maturation system on oocyte penetrability and male pronucleus formation and 3) to determine the ability of the in vitro matured oocytes to be fertilized in vivo by artificial insemination (AI) of sows. The differences examined between the 2 maturation systems included the culture medium (Waymouth vs TCM199), hormones, additives, culture conditions (static vs gentle agitation) presence or absence of porcine follicular fluid (PFF) and presence or absence of follicular shells. The results showed that nuclear maturation rate was similar in both systems (83.3 +/- 3.5 vs 86.4 +/- 2.5%), and intracellular content of glutathione was 5.21 +/- 0.73 vs 3.5 +/- 0.39 pmol/oocyte, although no correlation between these parameters was observed. The penetration rate and number of sperm cells per oocyte were dependent on the boar, maturation system and sperm concentration, but the rate of male pronuclear formation seemed to be influenced only by the boar and the maturation system but not by sperm concentration. In vivo fertilization of in vitro matured oocytes showed that both maturation systems could yield viable oocytes since 3 of 4 gilts and 2 of 4 gilts, respectively, became pregnant. Failure to become pregnant was not associated with inadequate oocyte maturation since control gilts, which received their own ovulated oocytes rather than in vitro matured oocytes at transfer, also did not become pregnant. We conclude that polyspermy may be an inherent problem in the IVF but not in the IVM systems.     

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.     

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.     

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.     

In vitro fertilization of in vitro matured canine oocytes using frozen-thawed dog semen

Experiments were conducted to evaluate in vitro fertilization (IVF) of in vitro matured (IVM) bitch oocytes using dog spermatozoa frozen in three different extenders. Sperm-rich fraction from eight ejaculates of five dogs was frozen in each one of three egg yolk Tris extenders with additional: (A) 1.4 g citric acid and 0.8 g glucose; (B) 0.7 g citric acid and 3.5 g glucose; or (C) 1.4 g citric acid and 0.8 g fructose (all with 5% glycerol in 100 mL milliQ water). Thawed sperm were co-incubated with IVM bitch oocytes for 6 h. Oocytes were fixed and evaluated under an epifluorescence microscope; penetrated oocytes were defined as those having sperm heads in the perivitelline space or in the oocyte cytoplasm. Higher penetration rates (P < 0.05) were obtained in oocytes cultured with spermatozoa frozen in extenders B and C than those frozen in extender A (33.1, 34.2 and 26.4%, respectively).     

Embryonic development of in vitro matured and in vitro fertilized dog oocytes

In several studies, early cleavage stage canine embryos have been derived from in vitro fertilized oocytes cultured under various conditions. Despite these results, IVF protocols for canine oocytes have yielded low fertilization rates. In this study, Experiment I compared the effects of tissue culture medium (TCM)-199 supplemented with either (A) 1 microg/ml estradiol or (B) 20 microg/ml estradiol + 1 microg/ml human somatotropin (hST) on the in vitro nuclear maturation rate of canine oocytes. Meiotic progression to the metaphase I and II (MI/MII) stages at 72 hr of in vitro culture (IVC) was 10.2% (11/108) in medium A versus 14.1% (30/142) in medium B (P = 0.802). In Experiment II, cleavage rate was determined among oocytes recovered from ovaries of bitches at different reproductive stages. Oocytes (n = 888) were retrieved from bitches at the follicular, anestrous, and luteal stages and selected for high morphological quality. Oocytes were matured for 48 hr in TCM-199 supplemented with 1 microg/ml hST + 20 microg/ml estradiol. Oocytes were in vitro fertilized with fresh canine spermatozoa that had been isolated on a Percoll gradient, and were cultured in synthetic oviduct fluid (SOF) medium with bovine serum albumin (BSA; 4 mg/ml) up to 5 days in 5% CO(2) in air at 37 degrees C. A proportion of oocytes (30.6%) with identifiable nuclear material had cytoplasm penetrated or fertilized by sperm. The percentage of oocytes developing into early stage embryos was 10.1% (27/267). Although pronuclear development was observed to be higher for oocytes recovered at the follicular phase, the cleavage rate was similar among oocytes recovered from bitches at the follicular, anestrus, and luteal stages. There was no correlation between the proportion of capacitated or acrosome reacted spermatozoa and pronuclei formation and/or percent cleavage. It was concluded that TCM-199 supplemented with 1 microg/ml hST and estradiol (20 microg/ml) supports nuclear and cytoplasmic maturation of canine oocytes. In this study, meiotic competence was verified by the in vitro production (IVP) and development of embryos up to the 8 cell-stage. Furthermore, the results indicate that, under the described conditions and despite the influence of reproductive status of the bitch on the developmental competence of in vitro fertilized oocytes to the pronuclei stage, cleavage was independent of donor's reproductive estrous cycle stage.     

Pentoxifylline improves in vitro fertilization and subsequent development of bovine oocytes

The purpose of this study was to examine whether pentoxifylline improves in vitro fertilization and developmental rates of bovine oocytes. In the first experiment, we examined the effects on the fertilization rate of various concentrations of pentoxifylline (0-7.5 mM) combined with heparin (10 IU/mL). In the second experiment, we examined fertilization cleavage and blastocyst rates after frozen-thawed spermatozoa, obtained from four different bulls, were incubated with heparin (10 IU/mL) with or without caffeine (5 mM) or pentoxifylline (5 mM). In the first experiment, a significantly higher fertilization rate was obtained in heparin containing 5 mM pentoxifylline compared to that in heparin alone or in heparin containing 7.5 mM pentoxifylline (86% vs 60% vs 64%, respectively). The percentage of monospermy in 5 mM pentoxifylline (81%) was significantly higher than in heparin alone (57%). In the second experiment, the interactions among Bulls A, B, C, and D; between treatments (pentoxifylline-with-heparin, caffeine-with-heparin and heparin alone), and between bulls and treatments were analyzed for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts. Among bulls, there was a significant difference in the number of oocytes penetrated (P < 0.01), monospermic oocytes (P < 0.05), cleaved oocytes (P < 0.001), and blastocysts (P < 0.001). Between treatments, there was a significant difference in the number of oocytes penetrated (P < 0.001), monospermic oocytes (P < 0.01) and cleaved oocytes (P < 0.001). Interaction between bulls and treatments was observed for the number of oocytes penetrated (P < 0.05). Individually, for Bulls A, C and D, the numbers of oocytes penetrated and monospermic oocytes in pentoxifylline-with-heparin were significantly higher than in heparin alone. For Bull D, significantly higher results were obtained for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts in pentoxifylline-with-heparin compared to caffeine-with-heparin and heparin alone (P < 0.05). These results suggest that treating sperm with 5 mM pentoxifylline in combination with heparin is effective for bovine in vitro fertilization and it that this treatment is effective even for bulls that produce low fertilization and blastocysts after sperm treatment with caffeine-with-heparin or heparin alone.     

Antioxidant requirements for bovine oocytes varies during in vitro maturation,fertilization and development

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.     

Maturation and sperm penetration of canine ovarian oocytes in vitro.

Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were in the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or II by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24--72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20% of those which had been cultured for 24--72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured in vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

In vitro fertilization, development, and implantation after exposure of mature mouse oocytes to visible light.

Mature mouse oocytes were exposed prior to in vitro fertilization to visible light during 1, 2, or 4 hr at an intensity of 4,000 lux. Compared to controls cultured under identical conditions but protected from light, exposed eggs did not show any significant modification of cleavage speed and rate. After transfer of blastocysts obtained in vitro in uteri of pseudopregnant females, the implantation rate and the proportion of normal fetuses were not found to be different in relation to preliminary light exposure of oocytes fertilized and cultured in vitro.     

Maturation and fertilization of porcine oocytes from primordial follicles by a combination of xenografting and in vitro culture

Our objective was to develop a method of endowing oocytes from porcine primordial follicles with full maturation and fertilizing ability as a model for ovarian xenografting of large mammals. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the capsules of both kidneys of ovariectomized athymic mice. The host mice were treated with 5 IU of equine chorionic gonadotropin (eCG) for 10 days (eCG-10), 30 days (eCG-30), or 60 days (eCG-60) after detection of cornified epithelial cells in their vaginal smears. Cumulus-oocyte complexes, ovarian grafts, and blood samples were obtained 48 h after eCG treatment. Forty-five to 70 days after grafting, the host mice in all groups for the first time showed vaginal cornification, accompanied by the formation of a small number of antral follicles in the grafts. However, we recovered large numbers of full-sized oocytes only from mice in the eCG-60 group; the numbers of full-sized oocytes in the other groups were low. Peripheral levels of total inhibin were highest in the eCG-60 group; this supports our finding that the most enhanced growth of antral follicles occurred in this eCG-60 group. Of 573 oocytes obtained from the eCG-60 group, 98 (17%) were at the metaphase II stage after in vitro culture for maturation. Moreover, 55% of matured oocytes with the first polar body (n = 20) were fertilized in vitro. These results clearly demonstrate that fertilization of oocytes from porcine primordial follicles is achievable by a combination of xenografting and in vitro culture.     

Maturation and fertilization of pig follicular oocytes cultured in pig amniotic fluid

Three experiments were conducted to assess the ability of pig amniotic fluid to support oocyte maturation and developmental competence. In Experiment 1, pig follicular oocytes were cultured in pig amniotic fluid (PAF) containing luteinizing hormone (LH) and estradiol-17beta for 28 to 48 h, during which time the maturational stages of the oocytes were observed. While the maturation rates to the second metaphase were high (62%) after 33 h of culture, the rates decreased (24 to 30%) when oocytes were cultured in PAF without LH. In Experiment 2, oocytes matured in PAF were inseminated in vitro with fresh semen from three boars. The spermatozoal penetration rate ranged from 42 to 100%, and 15 to 40% of the penetrated oocytes had both male and female pronuclei. In Experiment 3, oocytes were cultured for 46 to 47 h in PAF and transferred to the oviducts of inseminated gilts. Eighteen of 136 embryos recovered from the uteri 128 h after oocyte transfer developed to the morula and blastocyst stages. The embryos were transferred to a recipient, and two piglets were born (one live and one dead) of the resultant pregnancy. These results indicate that PAF can be used for maturation of pig follicular oocytes and that oocytes cultured in PAF have the developmental capacity to become piglets.     

In vitro fertilization of pig oocytes matured in vitro

The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy.