Vitrification, warming in sucrose-free medium and transfer of goat and mouse embryos
von Denisse Melissa Garza Hernández
Datum der mündl. Prüfung:2016-05-12
Betreuer:Prof. Dr. Dr. Matthias Gauly
Gutachter:Prof. Dr. Wolfgang Holtz
Gutachter:Prof. Dr. Christoph Knorr
EnglischIn farm animals, the usage of assisted reproduction programs, implying biotech-nologies such as estrus synchronization, superovulation, cryopreservation of embryos and embryo transfer, to mention some, helps to improve productivity and genetic value of the flock. The general objective of this investigation was the establishment of suita-ble means for embryo vitrification and embryo transfer that are easier to employ in as-sisted reproduction programs under farm conditions of small ruminants than existing methods. The objective was addressed in three separate trials: 1. Assessment of the survival rate of murine embryos after vitrification by an open or closed system and one-step warming in two different warming media. 2. Evaluation of the efficiency of two vitrification systems and one-step warming in sucrose free medium for the cryopreservation of goat embryos. 3. Comparison of the effectiveness of semi-laparoscopical versus transcervical em-bryo transfer in goats. The first experiment compares two different vitrification systems and two differ-ent warming solutions in mouse embryos. Cryopreservation of embryos is of considera-ble relevance for the implementation of embryo transfer programs and the establish-ment of embryo banks in several mammalian species. Vitrification was performed using “Open Pulled Straw (OPS) or CVM RingFibre plug™ (CVM) devices. Warming was car-ried out either in a warming solution containing 0.33 M sucrose or in a solution devoid of sucrose. Differences between vitrification systems were not significant. Warming in sucrose-containing diluent resulted in an expansion rate of 64%, as compared to 86% in a solution devoid of sucrose; reported hatching rates were 45% vs. 9%, respectively (p<0.05). Upon transfer, implantation rates for OPS- and CVM were 50% and 27%, re-spectively, compared with 55% for freshly collected embryos. The implantation rate after warming was 43% for sucrose-containing and 33% for sucrose-free medium. In conclusion: a) both vitrification systems are suitable for vitrifying mouse blastocysts; b) warming in sucrose-free diluent yields better embryo survival rates than in diluent containing 0.33 M sucrose. The second experiment outlines ways of non-surgical collection and semi-laparoscopic transfer of caprine embryos. Two different ways of embryo cryo-preservation by way of vitrification are described; the open pulled straw (OPS) proce-dure, known to be well suited and the solid surface procedure called for in situations where contact between embryos and non-sterile liquid nitrogen is to be avoided. Based on 13 transfers of OPS-vitrified and 9 transfers of solid surface-vitrified blastocysts (2 blastocysts/recipient) it was shown that either procedure is applicable (54% vs. 56% pregnancy- and 39% vs. 44% kidding rate). Furthermore the experiment showed that warming of vitrified embryos may be accomplished by one-step procedure (88% trans-ferable post-warming embryos), opening up the possibility to transfer vitrified embryos under field conditions. The third experiment consisted of an attempt to replace the semi- laparoscopic embryo transfer commonly practiced in our group by a noninvasive transcervical trans-fer technique. Pluriparous Boer goats (n=31) served as recipients. were submitted to an estrus synchronization protocol during the breeding season, consisting of insertion of a progestogen-containing CIDR for 7 days, followed, upon withdrawal 7 days later, by two doses of 5 mg dinoprost, applied at 12h interval. Does in estrus where considered suitable recipients and embryo transfer was carried out six days after the last day of standing estrus. For semi-laparoscopic embryo transfer does (n=22) were anesthetized and positioned in dorsal recumbency. Ovaries where laparoscopically inspected to lo-calize the ovary with at least one corpus luteum. With the aid of a blunted uterine te-naculum forceps (Pozzi; Aesculap, Germany), 255mm long, introduce via a 20 to 30 mm incision along the linea alba cranial to the udder, the tip of the uterine horn ipsilat-eral to the ovary displaying a corpus luteum was grasped under laparoscopic control. A loop of 20 to 30 mm of uterine horn close to the utero-tubal junction was gently exteri-orized. A puncture hole was made with a blunted 22 g hypodermic needle about 50 mm from the utero-tubal junction, through which a 20 µl unopette was introduced to deposit two embryos in the uterine lumen. Recipients received randomly selected embryos vitri-fied either with OPS (13 does) or CVM (9 does). For the non-surgical transcervical transfer nine pluriparous Boer goats served as recipients. To immobilize the does and prevent them from squatting, they were placed in a crate equipped with a hammock with holes for the front legs. A duck-bill speculum was introduced into the vagina, the os cervix was located and the lip of the os cervix was grasped with the aid of sharp –pointed uterine tenaculum forceps (255mm long) and carefully pulled caudally until it almost reached the vulvar orifice. A transfer catheter set for human embryo transfer, consisting of an atraumatic outer curved guiding cannula was introduce through the cervical canal and directed to the desired uterine horn (ipsilateral to the corpus luteum identified ultrasonographically). Recipients were randomly divided up, so four does received embryos vitrified with OPS and five does embryos vitrified by the CVM meth-od. Pregnancy was diagnosed by ultrasound 30 days and day 45 after transfer. Pregnan-cy rate for twenty-two does that received embryos semi-laparosocpically was 55% whereas, of nine does that received embryos via transcervical transfer only one re-mained pregnant. The semi- laparoscopic embryo transfer technique proved to be effec-tive, however still being a surgical procedure, entails anesthesia, a surgeon and aseptic environment, which are aspects not easy to maintain on-farm. Based on these results and previous trials by our work group, transcervical transfer of embryos could result in healthy born kids, however the pregnancy rates are very low and in order to get better outcomes the technique must be substantially improved.
Keywords: Vitrification; Warming; Goat embryos; Mouse embryos