Assisted reproduction technologies in goats

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Small Ruminant Research

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ssisted reproduction technologies in goats

aría-Teresa Paramio ∗, Dolors Izquierdoepartament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193 Bellaterrra, Barcelona, Spain

a r t i c l e i n f o

rticle history:eceived 11 June 2013eceived in revised form 4 November 2013ccepted 7 January 2014vailable online xxx

eywords:oatRTs

a b s t r a c t

Fertility in goats can be improved by natural methods (male effect and feeding) and byassisted reproductive technologies (ARTs). The main ARTs are: artificial insemination (AI),multiovulation and embryo transfer (MOET), in vitro embryo production (IVEP) and embryocryopreservation. Nowadays, somatic cell nuclear transfer (SCNT) is the methodology tocarry out reproductive cloning. Hormonal treatment to control and synchronize estrus andovulation is an essential methodology to implementing the rest of the ARTs. In goats, AI,MOET and IVEP are used to increase goat reproductive efficiency, genetic improvement,transport genetic material and to preserve genetic resources for possible use in the future.Goats have been the principal domestic animal of choice for biomedical research especially

mbryos for production of recombinant proteins secreted in milk. In recent years several studieshave been carried out on transgenesis and cloned goats. However, ARTs need to be appliedin healthy individuals to improve reproductive parameters. Poor animal health status, mal-nutrition and miss-management lie behind a reduction of fertility in spite of the techniquesused.

. Introduction

To improve fertility in goats 2 main strategies haveeen developed. The first one is based on natural methodsuch as “Male Effect” to induce and synchronize ovula-ion and the use of “programmed feeding” to improveamete production, embryo survival, foetal programmingnd colostrum production (see review Martin et al., 2004).he second strategy is the utilization of assisted repro-uction technologies (ARTs). These technologies involvestrus and ovulation control, artificial insemination (AI),ultiovulation and embryo transfer (MOET), in vitro

mbryo production (IVEP), cryoconservation of gametesnd embryos and somatic cell nuclear transfer (SCNT) orhat is colloquially referred to as “cloning” and its use in

Please cite this article in press as: Paramio, M.-T., Izquierdo,Ruminant Res. (2014), http://dx.doi.org/10.1016/j.smallrumres

he production of transgenic goats (pharming activity).In goats, artificial insemination is the most well-known

nd used reproductive technology. Commercial activity

∗ Corresponding author. Tel.: +34 935811456.E-mail address: [email protected] (M.-T. Paramio).

921-4488/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.smallrumres.2014.01.002

© 2014 Elsevier B.V. All rights reserved.

with goat embryos, both produced in vivo and in vitro, isless frequent compared to other ruminant species. How-ever, there are an increasing number of studies on cloningbecause of the growing interest in goats as animals express-ing recombinant therapeutic proteins in milk. Studies inSCNT are also important in improving oocyte quality andoocyte competence. Oocyte quality is used as synonymousof oocyte competence and defined as the ability of an oocyteto resume meiosis, cleave following fertilization, develop tothe blastocyst stage, induce a pregnancy and bring the off-spring to term in good health. Studies in SCNT will help toimprove our knowledge of embryos and gametes.

2. Artificial insemination

Artificial insemination has an important role in goatbreeding, especially in intensive systems of production,

D., Assisted reproduction technologies in goats. Small.2014.01.002

to control reproduction and in conjunction with accurateprogeny testing to improve the production of milk, hair andmeat. Buck ejaculates are small (1 mL) in volume with ahigh concentration of spermatozoa (4 × 109). The semen

dx.doi.org/10.1016/j.smallrumres.2014.01.002


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is extended in ultrahigh-temperature bovine skim milkto achieve a dose of 200 × 106 sperm per goat diluted in0.1 mL. Buck semen contains phospholipase A, an enzyme(BUS) secreted by the bulbo-urethral gland, that can trans-form phosphatidylcholines of egg yolk into lysolecithin,a toxic compound for spermatozoa. Milk proteins, espe-cially caseins and lactoglobulin, were also found to interactwith BUS to cause sperm deterioration (see review Leboeufet al., 1998). As a consequence, diluents for goat semenhave either been based upon skimmed milk or, alterna-tively, the seminal plasma has to be removed before usingegg yolk based diluents. Recently, some observers indi-cate that low levels of egg yolk (∼2.5%) may avoid toxiceffects (see review Cseh et al., 2012). The use of frozensemen overcomes farm dispersion problems, enhancingthe more accurate genetic evaluation of animals. However,the success of AI with frozen–thawed semen is lower thanwith fresh or cooled semen or that achieved by naturalservice. In Majorera breed, Batista-Arteaga et al. (2011)observed a kidding rate of 70% and 46% for fresh andfrozen semen, respectively. Overall AI fertility rates withfrozen–thawed semen vary from 40% to 65%, and seem tobe highly affected by factors such as season, farm (Leboeufet al., 2000; Salvador et al., 2005), reproductive status offemale (Dorado et al., 2007), and site of semen deposi-tion (Salvador et al., 2005). Studies in goats indicated thatwhen frozen–thawed semen was used increased depth ofsemen deposition increased pregnancy rate. The anatomyof the cervix in goats (a muscular canal with several cer-vical folds or rings) makes passage of the inseminationcatheter into the uterus difficult. In Murciano-Granadinabreed goats, trans-cervical insemination could be attainedonly in 17.5% of females and pregnancy rates improvedfrom 37% to 82% when frozen–thawed semen deposi-tion was post-cervical rather than vaginal (Salvador et al.,2005). This research group also showed that a dose of200 IU of oxytocin increased depth of semen depositionbut neither the kidding rate nor prolificacy was improved(Viudes-De-Castro et al., 2009). In conclusion, as the semenis deposited deeper in the genital tract, a higher rateof pregnancy is obtained, greater still when the catheterreaches the uterus. This seems to be more conclusive whenfrozen–thawed semen is used in comparison with chilledsemen and no effect was observed with fresh semen inMurciano-Granadina goats (Roca et al., 1997). The timingbetween semen deposition and ovulation is an importantfactor in the results of AI. Hormonal synchronization ofestrus and ovulation is based on progestagens, eCG (equineChorionic Gonadotrophin) and prostaglandins during thebreeding and non-breeding seasons. Currently, the mostcommonly used hormonal treatments involve the deliveryof a synthetic progestational agent (the most commonlyused commercial forms of progestagens are 20 mg fluoro-gestone acetate and 60 mg medroxyprogesterone acetateper sponge) via a vaginal sponge for 11 days (see reviewAbecia et al., 2012). Two days before sponge removal,the goats receive one injection of 250–600 IU eCG and


prostaglandin F2� (PGF2�). Artificial insemination is car-ried out 43–45 h after sponge removal. This treatmentpermits a conception rate of about 60–65% for out-of-season with frozen-thawed semen (Leboeuf et al., 2000).

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Other progestagens and other progestagen dispensers havebeen used in different countries (see review Fatet et al.,2011). Rubianes and Menchaca (2003) described a short-term protocol of 5–7 days with progestagen plus PGF2� andeCG resulting in synchronous estrus at approximately 30 hand ovulation 60 h after progesterone withdrawal. A preg-nancy rate of 64% was obtained when AI was performedat 54 h after device withdrawal (Menchaca et al., 2007).Vilarino et al. (2011) re-using intravaginal devices impreg-nated with 0.3 g of progesterone 3 times in a short-termprotocol found a pregnancy rates of 75%, 67% and 62% forfirst, second, and third use, respectively.

3. Embryos produced in vivo and in vitro

Commercial embryo transfer activity is not as impor-tant in goats compared to the cattle and sheep. Accordingto the data of the International Embryo Association (IETSNewsletter, December 2011) 1869 in vivo produced caprineembryos were produced worldwide, of which Australiaproduced 1200. For the same year 30,000 sheep embryosand 600,000 cattle embryos were produced in vivo plus340,000 produced in vitro. The total number of bovineembryos increased 10.3% compared to 2009.

In vivo embryos are obtained by MOET techniques andin vitro embryos by in vitro fertilization (IVF) and intracyto-plasmic sperm injection (ICSI). These embryo technologiestogether with the AI are the keys to improving the geneticstructure of animals. According to the bibliography thesetechnologies are studied considerably less in goats than incattle and sheep.

The high variability of ovulation rate in response tohormonal treatments, the fertilization failure and the pre-mature regression of Corpora Luteum (CL) are the mainlimitations on the use of MOET in goats (Cognie et al., 2003).In vitro production of embryos has been improved in recentyears but blastocyst yield is still lower and more variablethan in cattle. In our previous review (Paramio, 2010) weconsidered the percentage of in vitro produced blastocyst(blastocysts/oocytes) 12% and 36% from oocytes recoveredfrom prepubertal and adult goats, respectively.

In goats, multiovulation protocols consist of a prolongedprogestagen priming (12–18 days), with FSH administeredtwice daily for 3–4 days, beginning between 1 and 3days before the end of the progestagen treatment. FSH isobtained from the pituitary of sheep and pigs. The dosesand the number of injections depend on commercial rec-ommendations but a standardized protocol in goats couldbe 200 mg of porcine FSH or 8.8 mg ovine FSH. A luteolyticdose of PGF2� or one of its analogues is normally givenwith the first FSH injection. A short protocol called “Day0 protocol” has been designed by Rubianes and Menchaca(2003) and consists in a progesterone treatment for 5–7days plus an injection of PGF2� and 200–300 IU eCG atthe time of progesterone withdrawal. A GnRH injectionis given 36 h after progesterone treatment. The FSH treat-ment is initiated 72–84 h after progesterone withdrawal;


exogenous ovine FSH is administered in 6 decreasing dosesand PGF2� is administered in 2 half doses at the sametime as the last two doses of FSH. A dose of GnRH is given24 h after the first PGF2� administration. Comparing Day 0


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rotocol to the traditional protocol (Menchaca et al., 2010)n improvement in ovarian response (14.3 and 10.7 CL peroat, respectively) and in transferable embryos (5.9 and 4.2,espectively) was reported. The high variability in ovar-an responses to FSH observed between goats (between

and 30 transferable embryos per donor) is due to theollicular population present at the initiation of hormonalreatment and this affects ovulation and embryo produc-ion rates (González-Bulnes et al., 2004). A more recenttudy (Monniaux et al., 2011) concluded that the num-er of 1–5 mm diameter follicles was significantly relatedo the ovulation rate and embryo production response toSH treatment and this follicle population was the majorontributor in circulating Anti-Mullerien hormone (AMH).hus, we can predict the capacity of a donor goat to pro-uce a high or low number of high-quality embryos by aingle blood measurement, before FSH treatment, of AMHuring either breeding or anestrous seasons.

To avoid follicular dominance and increasingvulation treatments such as GnRH antagonist androgesterone–estradiol treatments should be tested inoats. These approaches, with the addition of other strate-ies such as the use of GnRH to synchronize ovulation andntrauterine insemination, have reduced ovulation rateariability, early CL regression and fertilization failure inoats (Menchaca et al., 2010).

In goats embryo collection involves more aggressiverocedure than in cows. To collect embryos by laparo-omy uterine horns must be flushed with medium toetrieve the embryos at 6–8 days after insemination. Thisrocedure allows 2–3 collections per goat because post-perative adhesions are a frequent sequel, limiting theumber of possible collections. Laparoscopic embryo col-

ection is less invasive but still needs full anaesthesia. Holtz2005) describes a non-surgical procedure using a rigidatheter passed through the cervix with an embryo recov-ry rate of 60–80%.

Compared to MOET, IVEP allows us to obtain offspringrom non-fertile females, such as prepubertal, pregnant,actating or even death or slaughtered females. The methodf IVEP involves three mains steps: in vitro maturation ofocytes (IVM), in vitro fertilization of oocytes (IVF) withapacitated sperm and in vitro culture (IVC) of embryos upo blastocyst stage to be transferred to recipient females orryopreserved for future use.

In goats, in vitro culture conditions for IVM, IVF andVC of embryos are quite standardized. In vitro matu-ation medium is TCM 199 with hormones, serum andysteamine or other antioxidants. Also, Hamster Embryoulture Medium (HECM) has been tested as a good IVMedium (Soberano-Martínez et al., 2011). IVM oocytes are

ertilized with sperm capacitated with heparin in TALPedium. After 24 h of gametes co-incubation, presump-

ive zygotes are cultured in SOF medium with serum for days to reach the blastocyst stage. In general, in vitroroduced embryos exhibit lower survival rates than their

n vivo counterparts and this difference is higher when


erum is added to IVC media (see review Amiridis andseh, 2012). Regardless of the culture media conditions,he percentage of blastocysts produced is highly vari-ble between laboratories and experiments. One of the

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principal causes of this variation is the unknown and differ-ent source of oocytes especially if they come from ovariesrecovered at the slaughterhouse. Oocyte selection is animportant part of the IVEP protocol. It is well-known thatoocyte competence to develop up to blastocyst is closelyrelated to follicle and oocyte diameter in cattle (Gandolfiet al., 2005). In goats, we have observed the relationshipbetween oocyte diameter and blastocyst production afterIVF (Anguita et al., 2007) and ICSI (Jimenez-Macedo et al.,2007). In a later study we have obtained 20% of blastocystsfrom oocytes recovered from follicles larger than 3 mmcompared to 4% of blastocysts from oocytes from folli-cles smaller than 3 mm and these percentages were thesame as in adult females (Romaguera et al., 2010, 2011).Oocytes recovered by laparoscopic ovum pick-up (LOPU)have the advantage that oocytes are obtained by a selectedfollicle with a specific diameter and appearance. Also, thehealth, nutrition and physiological conditions of oocytedonor females are well known. In contrast, this informa-tion is unknown in ovaries recovered at the slaughterhouse.Thus, this heterogeneous pool of oocytes has to be treatedusing the same culture conditions with obviously unpre-dictable results in embryo development. In our laboratorywe have tested Brilliant Cresyl Blue (BCB) staining to clas-sify the oocytes into 2 groups: blue stained oocytes (BCB+)or full-grown oocytes and unstained oocytes (BCB−) orgrowing oocytes. We have tested this methodology in goats(Rodríguez-González et al., 2002), cattle (Pujol et al., 2004)and sheep (Catalá et al., 2011). In all our studies we foundthat BCB+ oocytes developed up to blastocyst stage betterthan BCB− oocytes. We have observed higher MPF (Mat-uration Promoting Factor) activity, mitochondrial activityand ATP production in BCB+ oocytes than in BCB− oocytes(Catalá et al., 2011).

Intracytoplasmic sperm injection (ICSI) is another tech-nique for producing in vitro embryos, consisting of themicroinjection of a single sperm across the membrane of ametaphase II oocyte leading to fertilization. This techniquebypasses the normal sperm selection and oocyte–sperminteraction during fertilization. A major application of thistechnique for animal production includes use of genet-ically important male gametes for procreating wild anddomestic animals. There are only a few studies on ICSIas a method of fertilization in goats and there is onlyone report of live offspring (Wang et al., 2003). In ourlaboratory, the protocol used by ICSI consists of placingone matured oocyte into a microdrop of 5 �L of injectionmedium (TCM199) covered with mineral oil. A small vol-ume (1 �L) of sperm suspension is added to another 5 �Ldrop with a 10% polyvinilpirrolidone (PVP) medium. Theinjection pipette has an inner diameter of 7–9 �m andthe holding pipette measures 20–30 �m. The spermato-zoon is expelled into the ooplasm with a minimum volumeof medium (<5 pL). Using fresh semen capacitated withheparin (50 �g/mL), the injected oocytes had to be acti-vated chemically (with ionomycin and 6-DMAP) to startoocyte cleavage. This activation protocol induced a high


percentage of parthenogenic embryos. A second protocolwas carried out to overcome parthenogenesis. After spermselection, spermatozoa were capacitated with high con-centrations of sperm capacitator compounds (heparin plus


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ionomycin). Blastocyst yield from ICSI-oocytes of prepu-bertal goats was 16% (Jimenez-Macedo et al., 2005, 2006).

Embryo cryopreservation is essential for large-scalecommercial use of embryos. Two methods are used, slowfreezing and vitrified freezing. In the slow freezing methodembryos have to be adequately dehydrated and slowlycooled to avoid intracellular ice crystal formation. In thismethod a cryoprotective agent (CPA) such as ethyleneglycol (EG) or glycerol is used at a low concentration. Vitri-fication uses a highly concentrated CPA solution and it iscooled ultra-rapidly causing the CPA solution to changefrom liquid to a “glass-like” state without ice crystal for-mation. Literature has shown, independently of the methodused, the rate of embryo survival following slow freezingor vitrification are generally similar. The pregnancy ratesfollowing transfer of frozen-thawed embryos from goats is60–70% and 30–40% for in vivo derived embryos and in vitroproduced embryos, respectively (see review Youngs, 2011).Gibbons et al. (2011) found a survival embryo rate of 63.6%of embryos produced in vivo and vitrified. In embryos pro-duced in vitro and vitrified we did not find differencesin survival results either for prepubertal (51.8%) or adult(40.7%) goats (Morató et al., 2011).

4. Cloning and transgenic goats

The methodology of cloning animals using somaticcell nuclear transfer (SCNT) follows the protocol used byWilmut et al. (1997) to produce Dolly, the sheep clonedfrom an adult animal. The main steps involved in the pro-cess of SCNT are: (1) oocyte enucleation to obtain thecytoplast, (2) culture of somatic cell or nuclear donor cellcalled karyoplast, (3) transfer of the karyoplast to the cyto-plast, (4) fusion and activation of the karyoplast–cytoplastcouplets and (5) culture of cloned embryos up to blasto-cyst stage before transfer to the recipients. The utilizationof SCNT as a reproductive technology is still inefficient. Incattle, the proportion of reconstructed 1-cell embryos thatdevelop to transferable quality blastocysts is comparableto that following IVEP (close to 40% in both cases). How-ever, only 13% of blastocyst resulted in calves delivered atfull term compared to 30–45% of IVF-embryos. Moreover,peri-natal and post-natal mortality rates with cloned off-spring are greater than normally expected, with only 64%of cloned calves surviving to weaning at three months ofage (see review Wells, 2005). In goats, SCNT presents ahigh embryo loss (as in calves) however, no signs of pla-cental abnormalities, Large Foetal Syndrome, respiratoryor cardiovascular dysfunction, organ dysplasia, high peri-natal mortality or abnormal postnatal development havebeen seen as was observed in calves (Wilmut et al., 2002).

The intense effort carried out on SCNT research is mostlydue to the importance of transgenic research. Goats havean exceptional potential to serve as bioreactors to producemilk containing recombinant proteins suitable for phar-macological use. Goats have a short generation intervalcompared to cattle, two- to three-fold greater volume of


milk production compared with sheep and a lower inci-dence of scrapie. All of these make goats a suitable animalfor transgenesis. Moreover, dairy goats are ideal for the pro-duction of therapeutic recombinant proteins because their


high concentrations of recombinant protein of 1–5 g/L inmilk allows for herds of transgenic goats of manageablesize which could easily yield 1–300 kg of purified productper year.

Since Ebert et al. (1991) produced the first transgenicgoat using direct microinjection of the DNA constructsinto the pronucleus of zygotes, which expressed in milkthe human longer acting tissue plasminogen activator(LAtPA) at a concentration of 1–3 mg/mL, this techniquehas been used in goats to produce several proteins, suchas human factor IX (Zhang et al., 1997) human antitrom-bine III (Edmunds, 1998), lysozyme (Maga et al., 2006),butyrylcholinesterase (Huang et al., 2007) and lactoferrin(Zhang et al., 2008). The overall efficiency of gen microin-jection is poor, around 1% or less of the injected zygotesgive birth to a transgenic kid (Boulanger et al., 2012). Morerecently transgenic goats have been produced by SCNT.Somatic cells have to be genetically modified before trans-fer to enucleated oocytes. Using this method transgenicgoats producing malaria antigen (Behboodi et al., 2005) andhuman acid beta-glucosidase (Zhang et al., 2010) have beenobtained. The overall efficiency of nuclear transfer in goatsaverages 2.6% (total live kids/embryos transferred), makingthis technology an attractive alternative to microinjection.The impact of the production of a recombinant proteinin milk on the physiology of lactating goats and suck-ling young is more related to the biological activity ofrecombinant protein than to its absolute level of secre-tion. In some cases, a detrimental effect was reported onthe lactation physiology. Expression levels above 1 g/L ofhuman butyryl-cholinesterase decreased milk productionand reduced milk fat and casein contents. Expression ofhuman lysozyme resulted in a different outcome, with noimpact on growth or reproductive traits and a potentialimprovement in the health status of the lactating goats andtheir offspring through increased antimicrobial milk prop-erties. Apart from gene pharming, production traits suchas growth rate, quantity and composition of milk, fibreproduction or disease resistance have been obtained bytransgenic goats. However, to date, transgenic goats gen-erated for the benefit of production efficiency have notbeen reported. So far the only recombinant protein pro-duced by farm animals approved by the European Agencyfor the Evaluation of Medicinal Products in 2006 and theFood and Drug Administration in 2009 was rHAT producedin goats (ATryn® by GTC Biotherapeutics, USA). No otherfarm animal or its products have been marketed.

Cloning by somatic cell nuclear transfer (SCNT) has alsobeen used to reproduce transgenic goats. Baguisi et al.(1999) obtained 3 cloned transgenic goats generated bynuclear transfer of foetal caprine somatic cells to in vivo-derived caprine oocytes. The foetal somatic cells came from35 to 40-day transgenic recombinant human antithrom-bin III (rhAT) foetuses. Analysis of the milk of one of thetransgenic cloned animals showed high-level production ofhuman antithrombin III, similar to the parental transgenicline. In March 2012, Blash et al. (2012) reported that the


3 cloned goats born were alive and in good health. Thesegoats expressing rHAT did not manifest any instances ofmastitis and the growth and reproductive parameters wereabsolutely normal.


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In conclusion, goats present a good potential as animalsor biotech proposals with interesting results as cloned andransgenic animals. Studies in these fields would help us tomprove our knowledge of oocyte and embryos allowing uso improve our results using ARTs.

onflicts of interest

The authors do not have any conflict of interest toeclare.

cknowledgment

This study was funded by a grant of Ministry of Economynd Innovation of Spain: AGL2011-23784.

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Assisted reproduction technologies in goats