Small Ruminant Research 89 (2010) 144–148 In vivo and in vitro embryo production in goatsଝ Departamento de Ciencia Animal y de los Alimentos, Facultad de Veterinaria, Universidad Autónoma de Barcelona, 08193 Bellaterra, Barcelona, Spain Assisted reproductive technologies (ART) such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET) have been used to increase reproductive efficiencyand accelerate genetic gain. The principal limitations of MOET are due to variable female response to hormonal treatment, fertilization failures and premature regression of Cor- pora luteum. The in vitro production (IVP) of embryos offers the possibility of overcoming MOET limitations. The method of IVP of embryos involves three main steps: in vitro matu- ration of oocytes (IVM), in vitro fertilization of oocytes (IVF) with capacitated sperm and in vitro culture (IVC) of embryos up to blastocyst stage. Recovering oocytes from live selectedfemales by laparoscopic ovum pick-up (LOPU) and breeding prepubertal females by juve-nile in vitro embryo technology (JIVET) will allow a greater production of valuable goats.
Also, IVP of goat embryos will provide an excellent source of embryos for basic researchon development biology and for commercial applications of transgenic and cloning tech-nologies. Different protocols of IVP of embryos have been used in goats. However oocytequality is the main factor for embryos reaching blastocyst stage from IVM/IVF/IVC oocytes.
One of the principal determinant factors in the results of blastocyst development is the ageof the oocyte donor females. In goats, oocytes from prepubertal and adult females do notshow differences in in vitro maturation and in vitro fertilization; however the percentageof oocytes reaching blastocyst stage ranges from 12 to 36% with oocytes from prepubertaland adult goats, respectively.
2009 Elsevier B.V. All rights reserved.
1. Introduction
ogy, it can be applied to allow extra genetic gain throughthe production of embryos obtained from selected females Two exciting developments in goat reproduction are in vivo embryo production or multiovulation embryo transfer In vitro embryo production technology also presents the (MOET) and in vitro embryo production (IVEP), the tech- following advantages: (i) a significant increase of embryos nologies for altering or manipulating genetic material to from high genetic value females because oocytes can be improve the genetic structure of animals. The reproductive recovered from prepubertal, pregnant and even dead or technology most commonly used to accelerate genetic gain slaughtered goats, (ii) provides an excellent source of low has been artificial insemination (AI). However, although cost embryos for basic research, embryo biotechnology MOET cannot replace AI as a routine reproductive technol- studies (nuclear transfer, transgenesis, embryo sexing andstem cells) and all kinds of embryo research which needhigh number of embryos for manipulation and (iii) usedas a strategy for the rescue of some endangered animalspecies by interspecific embryo transfer. Moreover, embryo ଝ This paper is part of the special issue entitled: Plenary papers of the cryopreservation allows the movement and marketing of 9th International Conference on Goats, Guest Edited by Jorge R. Kawas.
goat germplasm providing safe worldwide movement of 0921-4488/$ – see front matter 2009 Elsevier B.V. All rights reserved.
doi: M.T. Paramio / Small Ruminant Research 89 (2010) 144–148 2. In vivo embryo production
tilization of oocytes (IVF) with capacitated sperm and invitro culture (IVC) of embryos until blastocyst stage that In vivo embryo production in goats has been studied for can be transferred to recipient females or cryopreserved years; however the results are not conclusive. The variabil- ity of the hormonal treatment, fertilization failure and thepremature regression of Corpora luteum still needs to be Traditional superstimulatory protocols consist of a Collection of good-quality oocytes is the first step for in prolonged progestagen priming (12–18 days), with FSH administered twice daily for 3–4 days, beginning between1 and 3 days before the end of the progestagen treat- • Oocytes recovered from slaughtered animals: Oocytes are ment. On average 8–16 ovulations are generated, although liberated from the follicles by aspiration, slicing or follicle individual variability is immense Several dissection. In adult goat ovaries, conventionally, oocytes attempts have been made in order to reduce this labor- are recovered by follicle aspiration selecting follicles big- ger than 3 mm diameter. From prepubertal goat ovaries, find differences by substituting the last three of six FSH- slicing the ovary allows collection of more oocytes per injections by a single dose of 200 IU eCG. When fixed-time ovary than by follicle aspiration (6.05 and 1.27), but the insemination is intended ovulation has to be timed. This may be accomplished by injection of LH, human chori- • Oocytes recovered from live goats: The techniques used are onic gonadotropin (hCG) or a GnRH-agonist. the aspiration of follicles after surgical exposure of the ovary by laparotomy or through laparoscopic ovum pick- not an efficient means of synchronizing ovulation unless up (LOPU). In order to recover high number of oocytes, the preceded by treatment with a GnRH-antagonist. The antag- donor goats are estrus synchronized and stimulated with onist temporarily suppresses FSH- and LH-release and, thus prevents the emergence of a dominant follicle. Some strate- using a unique injection of 80 NIH-FSH-P1 and gies have focused on starting the superovulatory treatment 300 IU of eCG at 36 h prior to LOPU obtained an average at wave emergence (in the absence of a dominant follicle).
initiation ofsuperovulatory treatment concomitant with the first fol- Transvaginal ultrasound-guided aspiration (TUGA) licular wave emerging after ovulation (day 0). Soon after technique used in goats has been described by ovulation, wave 1 emerged and there was a homogeneous cohort of growing small follicles. At day 0, FSH treatmentis initiated with six decreasing doses given twice daily.
3.2. Effect of the age of the goat donors on oocyte quality Two half-doses of PGF2␣ were given concurrent with thefifth and sixth FSH treatments. To synchronize the ovula- Several studies have reported lower embryo compe- tion GnRH analogue was injected 24 h after the first PGF2␣ tence from oocytes of prepubertal than adult females treatment. Results of this protocol showed higher ovula- tion rates and embryo yield than the traditional protocol reported a lack of development up to the blastocyst stage in prepubertal goats. A percentage of blastocysts of 10% To collect embryos by laparotomy, uterine horns must be flushed with medium to retrieve the embryos at 6–8 2-month-old females obtained in a slaughterhouse and days after insemination. This procedure allows 2–3 col- lections per goat, because post-operative adhesions are a with oocytes collected by LOPU from 2- to 5-month-old frequent sequel, limiting the number of possible collec- tions. Laparoscopic embryo collection is less invasive and gest that only a small proportion of the oocytes recovered allows 7 collections Collection via cervix, from 2 to 3 mm diameter follicles can support embryonic development because the capacity to complete cytoplas- mic maturation develops beyond the acquisition of meiotic In a successful goat MOET program an average of 6–8 competence. Thus, they showed that the goat blastocyst transferable embryos per donor can be produced, how- production was 6% with oocytes from follicles of 2–3 mm, ever, many factors (including breed, age and nutrition) in adult goats. In prepubertal goats, the number of follicles contribute to the high variability of transferable embryos (range from 0 to 30 per donor) with 25–50% of the donors which means that follicles larger than 5 mm in diameter failing to produce embryos due to fertilization failure and are practically non-existent in these females. In prepuber- tal goat oocytes, the percentage of blastocysts obtained is lower than those obtained from oocytes of adult goats.
Thus, 36%, 3. In vitro embryo production
26% and of blastocysts fromadult goat oocytes.
The method of IVP of embryos involves three mains Studying the differences between oocytes from pre- steps: in vitro maturation of oocytes (IVM), in vitro fer- pubertal and adult goats, we have found a lower male M.T. Paramio / Small Ruminant Research 89 (2010) 144–148 Follicular fluid (FF) from non-atretic and large follicles (>4 mm) has been used as a compound of matura- Estrus goat serum and FF need to be tested before being integrated in a protocol of in vitro embryo productionbecause both compounds present high chemical varia-tions between samples. adult goat oocytes used a maturation medium with defined Several studies in different species have concluded that compounds. The IVM used is TCM199 supplemented with oocyte diameter is directly proportional to follicle diam- 10 mg/ml EGF and 100 ␮M cysteamine, with good results eter. Increase in follicle and oocyte diameters improve The addition of different thiol compounds (cystine, cysteine, cysteamine, glutathione, ␤-mercaepthanol) to ference in the percentage of blastocysts obtained from the IVM media improve embryo development, increases oocytes recovered from follicles of 2–3 mm (6%), follicles of intra-cytoplasmic glutathione concentration (GSH) and 3.1–5 mm (12%), follicles >5 mm (26%) and from ovulated protects cells from culture oxidative stress. In prepu- oocytes (41%). In prepubertal goats, most of the oocytes bertal goat oocytes, testing different thiol compounds, come from 2 to 3 mm diameter follicles. In a recent study, cysteamine has been the thiol which significantly we have tested the relationship between follicle diam- increased intra-cytoplasmic GSH and embryo development eter and oocyte competence (Romaguera and Paramio, unpublished data). In this study, in prepubertal goats, the 100 ␮M of cysteamine improved embryo yield in oocytes percentage of blastocysts obtained from follicles bigger and smaller than 3 mm was 18.5% and 3.85%, respectively. As was indicated previously, follicular aspiration is difficult in oocytes (DOs) of adult goats, restored the GSH level prepubertal goat ovaries. Thus, it is easier to select oocytes and developmental capacity of DOs with cysteamine and liberated by slicing, according to their diameter and cumu- lus morphology. In our previous laboratory studies oocytediameter and blastocyst development after IVF concluded that a higher blastocyst rate was obtained in oocytes largerthan 135 ␮m (12.5%) compared to oocytes of 125–135 ␮m Before fertilization, buck ejaculates need to be prepared to inseminate the oocytes. The first step is to select the most we did not find differences between these two oocytes cat- motile and viable spermatozoa from the whole fresh ejacu- egories, 11.1% and 15.9%, of blastocysts ( late or the frozen-thawed sperm. The principal techniques In both studies, oocytes of 110–125 ␮m diameter used to select spermatozoa are swim-up and centrifuga- were able to develop up to morulae but they did not reach tion in Percoll or Ficoll density gradient. Greater yields of blastocyst stage. Oocytes smaller than 110 ␮m were unable highly motile spermatozoa can be obtained by swim-up, to cleavage. Comparing prepubertal to adult goat oocytes, when compared to Ficoll or Percoll density gradient cen- trifugation, but no differences were observed in terms of lower embryo production and pregnancy rates in oocytes oocyte penetration and cleavage rate after IVF with fresh from prepubertal than from adult goats.
goat semen For frozen-thawed goatsemen, conventionally, motile spermatozoa are obtainedby centrifugation on a discontinuous Percoll gradient. Once the most viable and motile spermatozoa were selected, Embryo development is influenced by events occur- sperm capacitation is carried out in media supplemented ring during oocyte maturation. For successful IVM, oocytes with heat-inactivated estrus serum (20% with fresh semen, must undergo synchronically nuclear and cytoplasmic maturation. Immature goat oocytes are convention- report capacitation of frozen-thawed sperm using 10% ally matured in buffered TCM199 supplemented with (v/v) estrus sheep serum in SOF medium and 0.5 ␮g/ml l-glutamine, pyruvate, hormones (FSH, LH and 17␤ - of heparin during 1 h. In our laboratory, after fresh sperm selection by swim-up, the supernatant is recov- Maturation media are generally supplemented with ered and capacitated in mDM with 50 ␮g/ml heparin for 10–20% heat-treated serum. In goats, estrous goat serum (EGS) and estrus sheep serum (ESS) are routinely used After oocyte maturation and sperm capacitation, oocytes are transferred to microdrops of modified Tyrode’s ESS and fetal calf serum (FCS). In our laboratory, we have plemented with hypotaurine and glutathione. Different tested EGS (at different times of estrus), fetal calf serum fertilization media have been used by different authors: (FCS) and steer serum (SS) and did not find any significant differences on maturation and embryo production M.T. Paramio / Small Ruminant Research 89 (2010) 144–148 3.6. Intra-cytoplasmic sperm injection (ICSI) In anaerobiosis conditions, different media are used: Intra-cytoplasmic sperm injection has been introduced as an alternative to assisted reproduction technology, espe- cially in humans. A major application of this technique for animal production includes use of genetically impor- tant male gametes for procreating wild and domestic animals. Moreover, this technique can be used to extend development of adult goat oocytes, concluded that the the sperm vector for transgenic animal production and to development of zygotes in SOF medium resulted in higher use freeze-dried sperm for which spermatozoa motility is blastocyst yield than in coculture with GOEC monolayer not required. The first live kids have been obtained by (28% and 20% of blastocysts, respectively), but after embryo n our laboratory, the protocol used by ICSI vitrification the percentage of embryos obtained with consists in placing one matured oocyte into a microdrop GOEC significantly improved the rate of pregnancy and sur- of 5 ␮l of injection medium (TCM199) covered with min- vival of embryos giving normal gestation as well as the birth eral oil. A small volume (1 ␮l) of sperm suspension is added of healthy offspring. In our laboratory the culture medium to another 5 ␮l drop with a 10% polyvinilpirrolidone (PVP) used is SOF (1 ␮l/embryo) plus FCS (0.1 ␮l per embryo) medium. The injection pipette has an inner diameter of added 24 h after presumptive zygotes were placed in the 7–9 ␮m and the holding pipette measures 20–30 ␮m. The spermatozoon is expelled into the ooplasm with a mini-mum volume of medium (<5 pl). Using fresh semen and 4. Conclusions
capacitation with heparin (50 ␮g/ml), the injected oocyteshad to be activated chemically (with ionomycin and 6- Despite recent progress made in MOET methodologies DMAP) to start oocyte cleavage. This activation protocol more research is needed to know the response, in ovula- induced a high percentage of parthenogenic embryos. A tion rate and embryo recovery, to exogenous hormones second protocol was carried out to overcome parthenogen- according to the follicular state of the ovary. Knowledge esis. After sperm selection, sperm were capacitated with of the follicular status and its repercussions on molecular high concentrations of sperm capacitator compounds (hep- characteristics of oocytes will be the major challenge to arin plus ionomycin). Blastocyst yield from ICSI-oocytes of optimizing both MOET and IVEP methodologies. In recent years IVEP has improved significantly. One of the reasons is the important number of research teams working in this field around the world. However the already dramatic dif-ference in IVEP results between laboratories and within laboratories is mostly due to the unknown oocyte quality.
The optimization of IVEP procedures must be consolidated After 24 h post-insemination (IVF or ICSI), presumptive over deep and basic knowledge of the biological material zygotes are removed from the fertilization medium and we are using, mostly the oocytes, but also the spermato- placed in an embryo culture medium. Early goat embryos zoa. This knowledge will be fundamental in improving goat cultured in vitro fail to develop past the 8–16-cell stages in productivity but will also be vital for the production and traditional culture media. This block occurred around time propagation of transgenic and cloned animals.
of activation of the embryonic genome. Serum and cells areadded to the culture to avoid this block.
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