ANS 3319C Reproductive Physiology and Endocrinology Techniques for In-Vitro Embryo Production Objectives 1) To gain an understanding of the process of in-vitro embryo production in cattle and other domestic species. 2) To provide hands-on experience in the techniques involved in in-vitro embryo production including oocyte aspiration and collection, maturation, fertilization, and embryo culture. Application of In-Vitro Embryo Technologies to Domestic Animal Species 1) Production of embryos from genetically valuable animals that are either infertile or that are deceased. 2) An alternative means to produce embryos from valuable animals rather than using superovulation 3) An inexpensive method for producing embryos using abattoir-derived ovaries Overview Although the production of domestic animal embryos in vitro is still a relatively new technique in commercial and clinical settings, work on in vitro fertilization began as early as the 1930 s with rabbit oocytes. While these first attempts at in vitro fertilization were not successful, subsequent research in the late 1950 s led to the birth of rabbit pups produced using oocytes fertilized in vitro. In the late 1960 s, work with human oocytes led to the birth of the first baby, Louise Brown (Figure 1), following in vitro fertilization in the United Kingdom in 1978. Since this time the production of human preimplantation embryos in vitro has become a common treatment for infertility. It is estimated that over 100,000 babies have been born using this technique in the United States alone. In terms of in vitro embryo techniques in domestic animals, the first calf (Figure 1), lambs and pigs produced following in vitro fertilization were born in the early 1980 s and the live birth of a foal following in vitro fertilization was reported in the early 1990 s. The production of bovine embryos in vitro has been the most successful of all the domestic animal species. Research on this topic in the 1980 s led to improved in vitro maturation medium and also techniques for capacitation of sperm in vitro. In the 1990 s new developments led to improved embryo culture conditions such that perimplantation bovine embryos could be cultured to the blastocyst stage in vitro. Over the last 10-15 years, the production of bovine embryos in vitro for commercial use has increased significantly and now there are several hundred thousand bovine in vitro produced embryos transferred worldwide each year. The production of embryos in vitro of other domestic species, especially the pig and the horse, have been less successful and further research is necessary before these techniques can be applied efficiently in commercial settings. This handout was developed by former Animal Sciences graduate students Jeremy Block and Luiz Augusto (Guto) de Castro e Paula. Jeremy and Guto received their PhD in reproductive physiology in the Department of Animal Science at the University of Florida. This handout was prepared using material from Dr. Pete Hansen s web page on production of bovine embryos http://www.animal.ufl.edu/hansen/ivf/default.htm.
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 2 Although there is variation among different species in the exact procedures for in-vitro embryo production, in general the procedure involves 4 major steps: 1) the collection of immature oocytes, 2) the maturation of immature oocytes, 3) the fertilization of mature oocytes and 4) the culture of embryos. Each of these steps will be discussed in more detail in the subsequent sections. Figure 1. Shown is the first baby born following in vitro fertilization, Louise Brown, in 1978 (left panel) and first calf born following in vitro fertilization, named Virgil, in 1981 (right panel). Oocyte Collection Immature oocytes in the form of cumulus-oocyte complexes (COC; figure 3) can be collected from live donor animals or directly from ovaries obtained from a abattoir. There are 2 main ways in which oocytes can be collected from live donors, 1) surgically by laparotomy and aspiration using a syringe and needle (Figure 2) or 2) using transvaginal ovum-pick-up techniques as shown in Figure 2. In the case of ovaries obtained from a abattoir or a deceased donor animal, the ovaries are generally transported to the laboratory in physiological saline that contains some form of antibiotic to help prevent bacterial contamination at approximately 22-24 ºC. The best results are obtained when oocytes are collected within 4-6 hrs after slaughter. Oocytes can be collected from abattoir derived ovaries either by aspiration using a syringe and needle or by slashing the surface of the ovary with a scalpel blade and collection of oocytes into a beaker as shown in figure 3. Oocyte Maturation Following collection, cumulus-oocyte complexes are washed several times and then placed into maturation medium for a specified amount of time depending on the species (Table 1). This process is meant to mimic what occurs in-vivo following the LH surge.
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 3 Thus during this culture period, the oocyte will resume meiosis and arrest at metaphase II so that it is ready for fertilization. The COC also undergoes other morphological changes during maturation, including the expansion of the cumulus cells (Figure 3). In some cases, such as in humans and horses, it is more common to allow oocytes to mature in vivo and then collect them for in vitro fertilization and embryo culture. Figure 2. Oocyte collection using ultrasound-guided transvaginal aspiration (left panel) and using a syringe and needle (right panel). Figure 3. Collection of oocytes by slashing the ovarian surface (left panel) and several immature bovine cumulus-oocyte complexes following collection (right panel). Although the mammalian oocyte resumes meiosis immediately after removal from the follicular environment it is important to place the COC into maturation medium as soon as possible following collection to provide an optimal microenvironment for oocyte maturation. The typical maturation medium will include several components including nutrients (pyruvate, glucose, glutamine, serum), hormones and gonadotrophins (estrogen, LH, FSH), and antibiotics (penicillin/streptomycin or gentamicin).
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 4 Species Cow Pig Duration of oocyte maturation 21-24 hrs 40-44 hrs Horse 24-48 hrs Human 28-36 hrs Mouse 16-17 hrs Table 1. Duration of in vitro oocyte maturation in various species. Collected COC are generally matured in 50 μl microdrops (10 COC/drop) or in wells of a 4-well plate (40-50 COC/well; Figure 4) overlaid with mineral oil to help prevent evaporation of the maturation medium. Once placed into maturation drops, COC are placed in an incubator set at 37-39 ºC for the desired amount of time depending on the species (Table 1). Figure 4. Petri dishes typically used for in vitro maturation (left panel) and mature bovine cumulus-oocyte complexes following 22-24 hrs of in vitro maturation (right panel). Note the expansion of the cumulus cells. In vitro fertilization For in vitro fertilization to occur, the media used must be capable of supplying the sperm cells with nutrients and chemical signals to enhance sperm motility and induction of capacitation, to facilitate the fusion of the gametes and the beginning of embryonic development. The in vitro fertilization process can be divided in three main steps: a. COC washing Necessary so that hormones, nutrients and metabolites present in the maturation microdrop are not carried over to the fertilization drop;
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 5 Procedure 1. Transfer COCs from each maturation microdrop to the X-plate containing the buffer HEPES-TALP; 2. Transfer 10 COCs from the X-plate to each well of the 4-well fertilization plate; b. Sperm purification Necessary so that sperm cells can be washed from the extender + criopreserver (if frozen is used)/seminal plasma (if fresh semen is used), selected for alive motile sperm cells and provided with nutrients, buffers and chemical signals to induce capacitation and hyperactivation. Procedure 1. Place 1.5 ml of 90% Percoll and 1.5 ml of HEPES-TL to one 15 ml conical tube; 2. Mix to make a solution of 45% Percoll; 3. In another 15 ml conical tube, add 3 ml of 90% Percoll; 4. Make a Percoll gradient (45% over 90%) by slowly layering the 90 % Percoll on the bottom of the tube containing the 45% Percoll using a plastic Pasteur pipet; 5. If using frozen semen (which is usually the case in IVF of domestic animal species), thaw enough straws of semen in a citothaw for 45-60 seconds or in another thermo with water pre-warmed to 37 o C; 6. Wipe the semen straw dry with a kimwipe, cut the tip of the straw with a scissors and expel contents of the straw onto the top of the Percoll gradient (Figure 5); Care must be taken so that the gradient is not disturbed and the semen lie on top of the 45% layer; Figure 5.. Layering of sperm onto Percoll. After cutting the tip of the straw (Left panel), the contents of the straw are expelled onto the top of the Percoll gradient (right panel). Here, removal of the semen is facilitated by using a homemade plunger.
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 6 7. Place the conical tube containing the semen and Percoll gradient into a centrifuge carrier that has been pre-warmed to 38.5 C, and centrifuge at 1000 x g for 10 min; 8. After centrifugation, collect sperm pellet from the bottom of the conical tube (Figure 6); Figure 6. Removal of sperm from the bottom of the Percoll gradient. 9. Place the sperm pellet into a 15 ml conical tube containing 10 ml Sp-TALP and place in a warm centrifuge carrier before centrifuging for 5 min at 200 x g; 10. Remove the supernatant with a Pasteur pipet while being careful not to disturb the pellet (Figure 7); Figure 7. Washing sperm in Sp-TALP. The left panel shows the washed and centrifuged sperm. The right panel shows the pellet of sperm remaining in the tube after aspiration of the supernatant.
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 7 11. Determine dilution required to bring sperm to a concentration of 26 x 10 6 /ml (this will produce a final concentration of sperm in the fertilization drop of 1 x 10 6 /ml) using a hemocytometer. Use IVF-TALP media to dilute the sperm solution (Usually 2 ml of IVF-TALP is enough to bring the content of 3 semen straws to the desired 26 x 10 6 sperm cells/ml); c. Fertilization At this point, sperm cells can be added to the wells containing the COCs so that fertilization can take place. Procedure 1. Add 25 µl sperm preparation (the IVF-TALP contains heparin which will help in capacitation of the sperm cells); 2. 25 µl PHE mix into each well (PHE is the acronym for penicillamine, hypotaurine and epinephrine which are molecules know to induce sperm hyperactivation); 3. Place the 4-well fertilization plates in a incubator (5% CO 2 in air) at 38.5 o C for 8-20 h; Alternative in vitro fertilization technique: Intracytoplasmatic sperm injection (ICSI) In certain circumstances, fertilization can be accomplished using ICSI. ICSI is widely used in humans when the male has poor sperm quality (low concentration, motility etc.), although, in some clinics, this is a standard procedure regardless of sperm quality. In this techinique, fertilization is assisted by injecting one selected sperm cell into one mature oocyte. ICSI is also generally used as the in vitro fertilization procedure in horses (See Figure 8). Figure 8. Immobilizing the sperm's tail before picking it up (left), injection of sperm into the egg (middle) and fertilized egg demonstrating the two nuclei--one from the father, one from the mother (right). Picutres from http://www.infertile.com/treatmnt/treats/icsi.htm.
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 8 Embryo culture Embryo culture is the step that follows fertilization. During this stage, the newly formed zygote will need to be provided with the conditions necessary to start dividing and grow in the most similar way to what would be expected to be happening in the first few days of pregnancy. There are several types of embryo culture: a. In vivo fertilized oocytes are placed in the oviduct of a synchronized female and grown for 6-8 days. The embryos are then retrieved and transferred to its definitive mother. In some cases, this type of culture can be performed across species, e.g., bovine or equine embryos can be successfully cultured in the sheep oviduct; b. Co-culture fertilized oocytes are placed in culture drops containing cells from the oviduct to try to mimic the maternal environment; c. Semi-defined medim fertilized oocytes are placed in culture drops containing serum. It is called semi-defined medium because the composition of the serum is variable and usually unknown. This is the most common method of embryo culture in domestic species; d. Defined medium fertilized oocytes are placed in culture drops where concentrations of all the components are known, including growth factors; e. Sequential culture the embryonic needs change as it grows. The idea of sequential culture is to place embryos in culture medium containing the nutrients necessary for one specific stage of development, mimicking the maternal environment; Two of the most common culture media used in embryo culture are SOF (Synthetic Oviduct Fluid) and KSOM Potassium Simplex Optimized Medium). Theses medias contain such items as nutrients (carbohydrates, amino acids) buffers, and antibiotics. Other factors affecting the embryonic development in vitro: a. Gas phase embryo culture can be done using determined concentration of gases. The two most commonly used are: 5% CO2 in air 5% CO2, 5% O2 and 90% N2. This gas mixture allows for better embryo development because it s thought to be more similar to the condition found in the oviduct and uterus b. ph the ph of the culture medium should be between 7.2 and 7.4. The ph can be affected by temperature and gas phase if not properly buffered; c. Osmolality should be between 260 280 mosm/kg; d. Temperature The culture temperature should be the same temperature found by the embryo in the oviduct and uterus. In the cow, the normal temperature ranges from 38.5 to 39 o C and so this temperature should used during culture; e. Water purity resistivity of water must be less than 18 mohm; f. Sterility procedures should be carried out taking care of keeping the culture sterile, using sterile techniques and antibiotics if necessary;
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 9 Bovine embryo culture procedure 1. Add 1000 μl HEPES-TALP in a microcentrifuge tube; 2. Place X-plate on the slide warmer and add ~5 ml of HEPES-TALP to each of the wells; 3. After microscope and air have been warmed sufficiently, remove one 4-well plate containing IVF drops from the incubator; 4. Remove oocyte-cumulus complexes (now called putative zygotes since many of them have been fertilized; Figure 9) from each well of the 4-well plate and place in the microcentrifuge tube. Up to 300 embryos can be loaded in one microcentrifuge tube; 5. Repeat steps 3 and 4 until all plates have been processed; 6. Remove cumulus cells from putative zygotes by vortexing (Figure 10) the tube containing the embryos/oocytes for 3-4 minutes; Figure 9. Putative zygotes after 8-hour fertilization. Note the cumulus cells detaching from the putative zygote due to action of the sperm cells. Figure 10. Vortexing COCs to remove cumulus cells. 7. Two to three days after fertilization, embryos should be checked under a microscope to determine the percentage that cleaved. This number is an approximation of the percentage of oocytes that were fertilized. However it should be noted that not only fertilized oocyte will cleave. Parthenotes are unfertilized oocytes that due to external stimulus start to divide. The parthenotes can reach blastocyst stage and when transferred to a cow, may survive for up to 30 days but will die due to failure in placentation and attachment to the uterus. 8. Seven or eight days after fertilization, embryos should be checked again and the percentage of blastocysts formed recorded. 9. Grade 1 and grade 2 blastocysts are usually transferred to synchronized recipients;
ANS 3319C Reproductive Physiology & Endocrinology Techniques for In vitro Embryo Production 10 Embryo culture in other species: further considerations The growth rate of the embryo varies among species being very fast in the murine and slower in the bovine.. Figure 11. Four-cell (top left) and 8-cell (top right) bovine embryos (cell s cytoplasms are seen in red and nuclei in blue). Bovine blastocyst on day 8 after fertilization (bottom). Inner cell mass is seen as a compact group of cells and the trophoblast as more disperse cells Embryonic growth requirement in vitro also varies greatly among species. For instance, murine embryos can grow with minimum nutrients, if any at all, while other species like bovine, porcine, equine and humans are much more selective and sensitive to non-optimal conditions. In most domestic species, embryos are transferred to the recipients at the morula or blastocyst stage. In humans, however, is a common procedure to transfer the embryos earlier, like at the 2-cell, 4-cell or 8-cell stages. This is to avoid the negative effects that prolonged in vitro culture has on the embryo (growth arrest, improper genome activation, large offspring syndrome). The drawback of this procedure is the need to transfer multiple embryos to increase the chances of establishing pregnancy which increases the chance of causing a multiple pregnancy (twins, triplets, quadruplets or more).