Successful culture in vitro of sheep and cattle ova
TL;DR: This communication describes the successful culture of one-cell to eight-cell sheep ova and one- cell and eight- cell cattle ova to the morula and blastocyst stages and reports a high embryo survival after transfer of cultured Ova to recipient animals.
Abstract: Fertilized sheep and cattle ova have not been reported to develop readily during culture in vitro. Up to 60% of sheep morulae develop normally during culture (Moor & Cragle, 1971) but earlier cleavage stages undergo limited development (Hancock, 1963; Kraemer, 1966; Tervit & McDonald, 1969; Moore, 1970) and it has been suggested that there is a block to development in vitro at the eightto twelve-cell stage (Wintenberger, Dauzier & Thibault, 1953). Only the early cleavage stages of cattle ova have been cultured and these have not been reported to develop beyond the twenty-four-cell stage in vitro (Thibault, 1966; Brinster, 1968; Sreenan, 1968; Sreenan, Scanlon & Gordon, 1968). This communication describes the successful culture of one-cell to eight-cell sheep ova and one-cell and eight-cell cattle ova to the morula and blastocyst stages and reports a high embryo survival after transfer of cultured ova to recipient animals.
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TL;DR: In a randomized 2 X 2 factorial trial of human in vitro fertilization that compared the two media and culture under oil versus culture in loosely capped tubes, significantly more clinical pregnancies were obtained with HTF medium than with T6 medium.
823 citations
TL;DR: This study indicates that embryos develop in vivo under low oxygen concentrations, especially during the peri-implantation period, and has implications for investigations of embryo metabolism and for improving embryo development in vitro.
Abstract: Oxygen tension was measured using flexible polarographic microelectrodes within the oviductal and uterine lumen in rhesus monkeys (n = 9), golden hamsters (n = 21) and rabbits (n = 6), during the reproductive cycle (monkey), during oestrus and pseudopregnancy (hamsters, rabbits) and during pregnancy (hamsters). In general, oxygen tensions in each species were much less than half of atmospheric O2, ranging from high values of about 60 mm Hg (8.7% O2) in the rabbit oviduct, rabbit and hamster uterus, to as low as 11 mm Hg (1.5% O2) in the monkey uterus. Oxygen tensions did not vary significantly between left and right sides of the reproductive tracts (all species), nor between pregnant and pseudopregnant states nor between oviduct and uterus (hamsters). Differences owing to reproductive stage were found in the monkey oviduct, hamster oviduct and uterus, and rabbit uterus. Oxygen tensions were consistently very low (11-14 mm Hg) in the monkey uterus throughout the menstrual cycle. In hamsters and rabbits, intrauterine O2 decreased significantly at about the normal time of blastocyst formation and implantation, to 37 mm Hg (5.3% O2) and 24 mm Hg (3.5% O2), respectively. This study indicates that embryos develop in vivo under low oxygen concentrations, especially during the peri-implantation period. The data have implications for investigations of embryo metabolism and for improving embryo development in vitro.
696 citations
TL;DR: It is indicated that porcine zygotes can develop into blastocysts in a chemically defined medium and to full term by transfer to recipients after culture.
Abstract: We evaluated the in vitro development of porcine zygotes that were cultured in a novel culture medium, porcine zygote medium (PZM), under different conditions and compared to in vivo development. The viability of these zygotes to full term after culture was also evaluated by embryo transfer to recipients. Porcine single-cell zygotes were collected from gilts on Day 2 after hCG injection. Culture of zygotes in PZM containing 3 mg/ml of BSA (PZM-3) produced better results in terms of proportion of Day 6 blastocysts, Day 8 hatching rate, and numbers of inner cell mass (ICM) cells and total cells in Day 8 embryos than that in North Carolina State University (NCSU)-23 medium. In culture with PZM-3, embryo development was optimized in an atmosphere of 5% CO2:5% O2:90% N2 compared to 5% CO2 in air. The ICM and total cell numbers in Day 6 embryos cultured in PZM-3 or in PZM-3 in which BSA was replaced with 3 mg/ml of polyvinyl alcohol (PZM-4) were also greater than those of NCSU-23 but less than those developed in vivo. However, no difference was found in the ratio of ICM to total cells among embryos developed in PZM-3, PZM-4, or in vivo. When the Day 6 embryos that developed in PZM-4 (99 embryos) or in vivo (100 embryos) were each transferred into six recipients, no difference was found in the farrowing rate (83.3% for both treatments) and in the number of piglets born (33 and 42 piglets, respectively). Our results indicate that porcine zygotes can develop into blastocysts in a chemically defined medium and to full term by transfer to recipients after culture.
585 citations
Cites background from "Successful culture in vitro of shee..."
...These media have been useful for in vitro culture of preimplantation sheep and cattle [16] and for mouse and human [17] embryos, respectively....
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...For in vitro culture of preimplantation embryos, synthetic oviductal fluid [16] and human tubal fluid [17] media have been developed, which are based on the composition of sheep and human oviductal fluid, respectively....
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TL;DR: Investigation of sheep zygote development of amino acids, ammonium, vitamins, and culture of embryos in groups in Synthetic Oviduct Fluid medium supplemented with BSA found indirect evidence that ruminant embryos utilize amino acids to a greater extent than do rodent embryos.
Abstract: The aim of this study was to develop a serum-free culture system that could support high levels of cleavage and blastocyst formation from sheep zygotes developed in vitro. To this end, we investigated the effects on sheep zygote development of amino acids, ammonium, vitamins, and culture of embryos in groups in Synthetic Oviduct Fluid (SOF) medium supplemented with BSA (32 mg/ml). The inclusion of amino acids in the culture medium had no effect on the percentage of embryos arrested at the 8-16-cell stage when embryos were cultured singly in the same drop of medium for 6 days (43% in SOF; 41% in SOF+amino acids). However, in medium containing all Eagle's amino acids, replacing the culture medium every 48 h to alleviate ammonium toxicity significantly decreased the number of arrested embryos (6%; p < 0.05) and significantly increased blastocyst cell number (52 cells in SOF; 105 cells in SOF+amino acids; p < 0.01) and the number of embryos developing to the blastocyst stage (29% in SOF; 67% in SOF+amino acids; p < 0.05). When the medium was renewed every 48 h, nonessential amino acids and glutamine also significantly decreased the number of arrested embryos (p < 0.05). Culturing embryos singly or in groups in SOF medium with all Eagle's amino acids that was renewed every 48 h resulted in significant increases in blastocyst hatching and mean cell number (47%, 31%, and 79%; 105, 136, and 173 cells for embryos cultured singly, in groups of 2, and in groups of 4, respectively). After culture in groups of 4, blastocyst cell numbers were equivalent to in vivo-developed controls (160 cells) and significantly greater than those developed in serum (103 cells; p < 0.01). Analysis of blastocyst metabolism, expressed on a per-cell basis, revealed that amino acids did not affect either glucose uptake or lactate production, whereas the addition of amino acids and vitamins resulted in a significant increase in both parameters (p < 0.01). A similar response was observed in serum-derived blastocysts. Ammonium production by sheep blastocysts after culture in the presence of amino acids was significantly greater than that produced by mouse blastocysts, indirect evidence that ruminant embryos utilize amino acids to a greater extent than do rodent embryos.(ABSTRACT TRUNCATED AT 400 WORDS)
558 citations
TL;DR: It is demonstrated that a semi-chemically defined medium can successfully support the development of bovine embryos to the morula stage to a limited extent, but the medium lacks some nutrients or growth factors to fully support development through the blastocyst stage.
532 citations
References
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TL;DR: A method for relatively large-scale in vitro experiments on the early cleavage divisions of the mouse embryo has been developed in this laboratory and is now being used in several types of study.
424 citations
"Successful culture in vitro of shee..." refers methods in this paper
...The embryos were cultured in droplets of medium under light-weight liquid paraffin in an atmos¬ phere of 5% C02 in air (Brinster, 1963)....
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TL;DR: A technique for obtaining a high proportion of viable embryos after subjecting them to sub-zero temperatures is described.
Abstract: COMPARED with the successful storage of spermatozoa and other tissue cells at sub-zero temperatures, attempts to preserve frozen mammalian eggs have been disappointing (reviewed in refs. 1 and 2), although a few pregnancies have resulted from the transfer of previously frozen fertilized rabbit eggs3 and unfertilized mouse eggs4 to recipient foster mothers. I now wish to describe a technique for obtaining a high proportion of viable embryos after subjecting them to sub-zero temperatures.
416 citations
TL;DR: It is concluded that homologous serum is an unsuitable medium for egg transfer but that TCM 199 is highly satisfactory.
Abstract: Summary. A total of ninety-two heifers was used in a series of four experiments designed to investigate the effect of method of egg recovery, transfer, storage medium and degree of synchronization between donor and recipient compatible with fertility. Experiments 1 and 2 involved the transfer of thirty-three eggs in homologous serum to eighteen recipients. In half of these, the eggs were transferred surgically and in the other half non-surgically. No pregnancies resulted from these transfers. In Experiments 3 and 4, a total of seventy-six eggs was transferred to forty recipient heifers using TCM 199 as the transfer and storage medium. Twenty of these transfers were made surgically, the eggs also being recovered by surgical means in eleven cases and by slaughter in nine. The pregnancy rate obtained was 91% for surgically recovered and transferred eggs and 33% for eggs recovered at slaughter and then transferred surgically. The remaining twenty animals received eggs, half of which were recovered surgically and half at slaughter, and all then transferred by non-surgical means. It is concluded that homologous serum is an unsuitable medium for egg transfer but that TCM 199 is highly satisfactory. The recovery of eggs from slaughtered animals appears less satisfactory than if they are recovered in vivo. A degree of variation from exact synchronization of \m=+-\2days can be tolerated.
119 citations
TL;DR: The chemical composition of fluid from the fallopian tube of the ewe was studied in four cycling ewe groups and in four groups of four ovariectomized ewes receiving oestrogen and progesterone in factorial combination.
Abstract: The chemical composition of fluid from the fallopian tube of the ewe was studied in four cycling ewes and in four groups of four ovariectomized ewes receiving oestrogen and progesterone in factorial combination Both the major electrolytes and some organic constituents in the fluids were examined
97 citations
"Successful culture in vitro of shee..." refers background in this paper
...The results showed that ova cleaved most readily in a medium with a composition based on the biochemical analysis of sheep oviduct fluid (Restall & Wales, 1966)....
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