survivability of parthenogenetic embryos following in vivo transfer in naturally synchronized capra...

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Survivability of parthenogenetic embryos following in vivotransfer in naturally synchronized Capra hircus

Ravi Ranjan & Rakesh Kumar Singh & Thirupathi Yasotha & Manish Kumar &

Kuldeep Kumar & Renu Singh & Monzamul Houque & Vijay Prakash Mourya &

Gyanendra Singh & Mihir Sarkar & Bikash Chandra Das & Sadhan Bag

Received: 20 February 2013 /Accepted: 20 May 2013 /Published online: 6 June 2013 / Editor: J. Denry Sato# The Society for In Vitro Biology 2013

Abstract The present study was conducted to see the invivo developmental potency of caprine parthenogenetic em-bryos generated in a modified way. The good quality caprineoocytes were matured in presence of cytochalasin B (CCB)and then activated by 7% ethanol followed by 2 mM 6-dimethyl amino purine (6-DMAP) and embryo developmentwas recorded. Early stage parthenogenetic embryos (two tofour cells) were surgically transferred in recipients (10). Thepregnancy diagnosis was done by nonreturn to oestrus,ultrasonography (USG), and progesterone estimation. Thelevels of progesterone were above normal values (1 ng/ml)of pregnancy and fall below the level of pregnancy justbefore retuned to oestrus. Progesterone profile revealed thatout of ten recipients (G1–G10), four goats (G1, G2, G3, andG5) returned to oestrus after 43±7.29 (Mean±SE) d of

embryo transfer and six goats (G4, G6, G7, G8, G9, andG10) did not return to cycle even after 70 d of embryotransfer. In three recipients (G4, G5, and G6), the USG onday 40 revealed that there was fluid filled uterine body withsolid fetus-like structure. These might be dead fetus and hadstarted resorption. The progesterone profile also corroborat-ed the assumption of pregnancy in these animals. Authorsbelieve that this may be the first report on in vivo diploidparthenogenetic embryo development in caprine species.

Keywords Caprine . Parthenogenetic embryo . Embryotransfer . Developmental potency . Ultrasonography

Parthenogenesis is a common mode of reproduction in loweranimal like bees, flies, and many avian species. However,sporadic cases of live birth in higher animals like commododragon and bony head shark have been reported by thisprocess. Recently, parthenogenetic embryos have been gener-ated in domestic animals like pig (Kure-bayashi et al. 2000; Yiand Park 2004), sheep (Loi et al. 1998), bovine (Fukui et al.1992), goat (Ongeri et al. 2001; Pankaj et al. 2012), andbuffalo (Bianca et al. 2004; Sathisha 2009; Venkatesh 2009).The in vitro aswell as in vivo developmental potency (Boedionoand Suzuki 1994; Kono et al. 1996; Loi et al. 1998; Kawarasakiet al. 2009) of parthenogenetic embryos of some of theseanimals have also been done. It was observed that whenparthenogenetic embryos were transferred to surrogate moth-er, they were capable to surviving up to day 10 in mouse(Kono et al. 1996), day 21 in sheep (Loi et al. 1998), day 40 inpig (Kawarasaki et al. 2009), and day 67 in cattle (Boedionoand Suzuki 1994). In most of these cases, haploid or homo-zygous diploid embryos produced (Lagutina et al. 2004) weretransferred. However, no information is available on in vivosurvivability of parthenogenetic embryos in caprine species.

R. Ranjan : R. K. Singh : T. Yasotha :M. Kumar :K. Kumar :R. Singh :B. C. Das : S. BagReproductive Physiology and Embryo Transfer TechnologyLaboratory, Physiology and Climatology Division, Indian VeterinaryResearch Institute, Izatnagar, Uttar Pradesh 243 122, India

M. HouqueDivision of Surgery, Indian Veterinary Research Institute,Izatnagar, Uttar Pradesh 243 122, India

G. Singh :M. SarkarNuclear Research Laboratory, Physiology and ClimatologyDivision, Indian Veterinary Research Institute, Izatnagar, UttarPradesh 243 122, India

V. P. MouryaClimatology Laboratory, Physiology and Climatology Division, IndianVeterinary Research Institute, Izatnagar, Uttar Pradesh 243 122, India

R. Ranjan (*)Physiology, Reproduction and Shelter Management Division,Central Institute for Research on Goats, Makhdoom, Farah,Mathura, Uttar Pradesh, Indiae-mail: [email protected]

In Vitro Cell.Dev.Biol.—Animal (2013) 49:486–491DOI 10.1007/s11626-013-9643-z

It has been postulated that in mammal, parthenogeneticembryos fail to produce live birth because of haploidy andlack of paternal genomes (Young et al. 2003). CytochalasinB (CCB) has been shown to inhibit first polar body extru-sion creating diploid heterozygous parthenogenetic embryosin mice (Jacek et al. 1991) and this chemical can also beused for diploid parthenogenetic embryos in caprine also(Ranjan et al. 2013).

The present experiments were conducted to study thein vivo survivability of parthenogenetic embryos gener-ated in modified way by using CCB during oocytesmaturation following transfer in recipients in caprine(Capra hircus).

Ethics. All the experiments were approved by ethics com-mittee of Indian Veterinary Research Institute, Izatnagar,Bareilly, Uttar Pradesh, India.

Chemicals and media. All media and chemicals of wereprocured from M/S Sigma, St. Louis, MO, unless otherwisementioned.

Oocytes collection and processing. Goat ovaries were col-lected from the local slaughter house and transported tothe laboratory in normal saline solution fortified withgentamycin (10 μg/ml) in a thermo flask at 37°C within2 h of slaughter. Oocytes were aspirated from all visiblenon-atretic follicles by aspiration method using oocytecollection media. The cumulus oocyte complexes weresearched out, evaluated, graded, and finally dropwashed with maturation media (MM) (TCM-199 withHEPES media, 10% FBS, 10% goat follicular fluid,

0.5 μg/ml FSH, 10 μg/ml LH, 0.5 μg/ml estradiol;Ranjan et al. 2013).

In vitro maturation, activation, and culture of oocytes. Oocyteswere matured in MM having CCB (15ug/ml) for diploidparthenogenetic embryos generation (Ranjan et al. 2013).After 27 h of incubation, the extent of oocytes maturationwas evaluated based on the visual assessment of degree ofcumulus expansion under inverted microscope (Kobayashi etal. 1994). Matured oocytes activated by 7% ethanol for 5 minfollowed by incubationwith 2mMDMAP for 4 h then washedseveral times in modified synthetic oviductal fluid (mSOF)and cultured in 100 μl of same media in CO2 incubator at37°C, 5% CO2, and 95% relative humidity. The cleaved oo-cytes were subsequently transferred in fresh drop of mSOF forfurther embryo development. After 48 h of in vitro culture, thecleavage rate and further embryo development was recorded.

Surgical transfer of embryo. Parthenogenetic embryos (twoto four cells stage) were surgically transferred in ten oestrussynchronized recipient. Two parthenogenetic embryos wereplaced in uterine horn ipsilateral to corpus luteum.Following embryo transfer, two conjugative injection ofinsulin (0.2 IU/ Kg BW) were given to enhance conceptionrate. Schedule for oestrus synchronization and embryotransfer as stated below. Post operative care and manage-ment was done as per standard surgical procedures.

Day 0 11 13 15 17 18

PGF2 α PGF2 α Estrous Embryo Insulin Insulin

12.5 mg 12.5 mg cover Transfer

Table 1. Pregnancy diagnosis on the basis of nonreturn to oestrus by HD, ultrasonography, and progesterone profile after parthenogenetic embryotransfer in synchronized caprine

Goat no. Nonreturn days on thebasis of HDa

Ultrasonography resultb Pregnancy diagnosisbased on serum P4 level

G1 58 Never detected fetus-like structure Negative

G2 30 Non pregnant Negative

G3 31 Non pregnant Negative

G41 Nonreturned to estrusc Detected fetus-like structure on day 40 and negative on day 50 Positive

G51 53 Detected fetus-like structure on day 40 and negative on day 50 Positive

G61 Nonreturned to estrusc Detected fetus-like structure on day 40 and 50, but negative on day 60 Positive

G7 Nonreturned to estrusc Never detected fetus-like structure Negative




a Average nonreturn to estrus was 43.00±7.29 (mean±SE) daysb Ultrasonography started after 30 d of embryo transfer and repeated after 10-d interval till the confirmation of pregnancy status or return to cyclicityc These animal did not return to oestrus even after 70 d of embryo transfer1 In these recipients, we found fluid-filled uterine body with solid fetus-like structure. The progesterone profile also corroborated the pregnancy

SURVIVABILITY OF PARTHENOGENETIC EMBRYOS IN GOAT 487

Figure 1. Progesterone profileafter diploid parthenogeneticembryo transfer in caprine(X-axis- days of bloodcollection after diploidparthenogenetic embryotransfer and Y-axis -P4 ng/ml;G, goat no.; P, pregnant; NP,non-pregnant; RC, return tocyclicity; NRC, nonreturn tocyclicity; USG+VE,ultrasonography positive after30 d of embryo transfer;USG–VE, ultrasonographynegative after 30 d of embryotransfer).

488 RANJAN ET AL.

Monitoring of nonreturn of recipient by parading of teaserbucks. Heat detection (HD) of recipients was carried outafter 25 d of embryo transfer with teaser buck twice a dregularly.

Ultrasonography (USG). The ultrasonography was startedon day 30 of embryo transfer and repeated every 10 dinterval until return to oestrus or up to day 70 by usingreal-time ultrasonography machine (Hitachi-Aloka MedicalLtd. Model- SSD 500, Tamilnadu, India).

Progesterone assay. For progesterone assay, blood samplesof recipients were collected 10 d interval from the date ofoestrus till day 70 or return of cyclicity following embryotransfer. The serum progesterone concentration was estimat-ed by Radio Immune Assay with a standard diagnostic kit(Immunotech, A Beckman Coulter Co. ISO 9001, FranceRef- IM1188). The minimal detectable concentration ofprogesterone was 0.08 ng/ml and intra- and inter-assay co-efficient of variation were 5.8% and 9.4%, respectively.

Statistical analysis. The embryo development was recordedas percentage of cleaved oocytes. The data was analyzed byapplying one way ANOVA using the SPSS 16 computerprogram as different variables and results have beenpresented as Mean±SE.

The overall cleavage rate percentage was 77.22±1.04 byusing 7% ethanol and 2 mM DMAP as activating agent. Thepercentage of two to four cell stage embryos was 22.90±1.38 out of total cleaved oocyte and rest were 8–16 cell andmorula stage of parthenogenetic embryos after 48 h ofactivation. The in vivo survivability of parthenogenetic em-bryos has been presented in Table 1, Fig. 1, G1-10 andFig. 2. The in vivo embryos development was monitoredby nonreturn to oestrus, USG, and progesterone (P4) assayof individual recipients.

The results indicated that based on nonreturn tooestrus, four recipients (G1, G2, G3, and G5) returned

to cyclicity after 58, 30, 31, and 53 d of embryotransfer with a mean of 43.00±7.29 d. Out of thesefour recipients, fluid filled cavity with fetus-like struc-ture was detected on day 40 of USG in G5 recipient(Fig 2). Further, P4 level in this recipient was also quitehigh and continued until about day 55 of embryo trans-fer (Fig. 1, G5). Neither fetus-like structure could bedetected by USG, nor was serum P4 level higher inrecipients G1, G2, and G3 indicating that in theseanimals, the embryo did not survive (Fig. 1, G1–G3).

The remaining six recipients (G4, G6, G7, G8, G9, andG10) did not show cyclicity even after 70 d of embryostransfer. USG results indicated fetus-like structure in two(G4 and G6) of these recipients (Fig. 2). Serum P4 level inthese two recipients (G4 and G6) was also found very high(Fig. 2G4, G6) until days 60–65 of embryo transfer indicatingthat parthenogenetic embryos survived in these recipients. Inthe remaining recipients (G7, G8, G9, and G10), no fetus-likestructure was detected through USG even on day 70 and theserum P4 levels were also very low (Fig. 1 G7–G10) whichnever reached above 1 ng/ml signaling that the parthenoge-netic embryo did not survive following transfer.

The quest for producing parthenotes is enormous as thiscould be an alternative to cloning of an animal. With thisquest, parthenogenetic embryos have been generated andthe in vitro and in vivo developmental potency are beingstudied in different species. In caprine, parthenogeneticembryos have been generated and their in vitro develop-mental potency has also been studied (Ongeri et al. 2001;Pankaj et al. 2012; Ranjan et al. 2013). But no studies havebeen made on in vivo developmental potency of partheno-genetic embryos in this species.

In the earlier study cytochalasin B (CCB) was usedwith the idea of generating diploid parthenogenetic em-bryos and it was observed that about 83% parthenoge-netic embryos were diploid (Ranjan et al. 2013).Chemicals like CCB, is an actin polymerization inhibi-tor, shortens actin filaments by blocking monomer ad-dition at the fast-growing end of polymers and in thepresence of CCB, segregation of the chromosomes oc-curred, but cytokines is does not take place. Therefore,this chemical has been used to produce diploid zygotewith two pronuclear (Presicce and Yang 1994a, b; Liuand Yang 1998). In our earlier study, we have observedthat exposure of oocytes with CCB during maturationresulted in very high percentage of diploid parthenoge-netic embryos without compromising the cleavage rateand in vitro developmental potency (Ranjan et al. 2013).

The parthenogenetic embryos survived from 43 to65 d in three out of ten recipients. The serum proges-terone values remained higher than 1 ng/ml, i.e., 2 to3 ng/ml during early stage of pregnancy and diagnosisof pregnancy could be predicted accurately (100%) on

Figure 2. Ultrasonography report as revealed by real-time ultrasonog-raphy machine (G, goat no.). (USG was started on day 30 of embryotransfer and repeated every 10-d interval until return to oestrus or up to70 d).


the basis of serum progesterone values around 17–19 dpost-mating (Wani 1989). In indigenous small EastAfrican goats, the mean plasma progesterone concentra-tion has been reported from 2.6 to 10.8 ng/ml fromconception to the mid-gestation (Kanuya et al. 2000).

It was expected that in more number of animals, theparthenogenetic embryos would have been survived.The reasons for majority of parthenogenetic embryosshowing in vitro or in vivo developmental defect couldbe due to the absence of paternal set of chromosomesbecause of haploidy. We used CCB to generate hetero-zygous diploid parthenogenetic embryos and it was as-sumed that these embryos would have had normal invivo developmental potency. However, further study isneeded to devise protocol so that all the embryos pro-duced are diploid and retain the same developmentalpotency as IVF or in vivo-derived embryos.

Although some recipients returned to cyclicity, manydid not even after 70 d of surgical embryo transfer.USG and progesterone evaluation reflected that theseanimals indeed did not conceive (Fig. 1, G1, G2, G3,G7, G8, G9, G10). The exact reasons for this could notbe ascertained but it may be due to the effect ofsurgical intervention (laparotomy) for embryo transfer.Our diploid parthenogenetic embryos generation proto-col did not produce 100% diploid embryos. So, itshould be checked for ploidy before embryo transferas haploidy and mixploidy causes developmental defectdue to aberrant gene expression or chromosomal abnor-malities. The premature termination of conception de-layed next oestrus (Goel and Agrawal 1991) in goat andonly 26.31% goats came in to oestrus within 6 mo ofkidding or abortion indicating long postpartum intervalin Indian goats (Kharche et al. 2008). The results indi-cated that diploid parthenogenetic embryos can be gen-erated using CCB in modified way survived about 30–65 d in vivo in the recipients following embryo transfer.To the best our knowledge, this is the first report on invivo survivability of parthenogenetic embryos in thisspecies (Capra hircus)

Acknowledgments The authors are thankful to the director of IVRI,Izatnagar, for providing all necessary facilities to conduct this experiment.The authors are also thankful to NAIP, ICAR, for funding the project.

References

Bianca G.; Lucia B.; Anna De R.; Rosella D. P.; Guiseppe C.; Luigi Z.I. Chemical activation of buffalo oocytes by different methods.Theriogenology 62(9): 1627–1637; 2004.

Boediono A.; Suzuki T. Pregnancies after transfer of aggregated par-thenogenetic bovine activated oocytes. Theriogenology 41: 166;1994.

Fukui Y.; Sawai K.; Furudate M.; Sato N.; Iwasumi Y.; Ohzaki K.Parthenogenetic development of bovine oocytes treated with eth-anol and cytochalasin B after in vitro maturation. Mol. Reprod.Dev. 33: 357–362; 1992.

Goel A. K.; Agrawal K. P. Indication and synchronization of oestrus inanoestrus recipient goats. Indian J. Anim. Reprod. 12: 187–189;1991.

Jacek K.; Andras P.; Michele W.; Bernard M. Genetically identicalparthenogenetic mouse embryos produced by inhibition of thefirst meiotic cleavage with cytochalasin D. Development 111:763–769; 1991.

Kanuya N.; Kessy B. M.; Nkya R.; Mujuni P. F. Plasma progesteroneconcentrations and fertility of indigenous small East African goatsbred after treatment with cloprostenol. Small Ruminant Res. 35:157–161; 2000.

Kawarasaki T.; Otake M.; Tsuchiya S.; Shibata M.; Matsumoto K.;Isobe N. Co-transfer of parthenogenotes and single porcine em-bryos leads to full-term development of the embryos. Anim.Reprod. Sci. 112: 8–21; 2009.

Kharche S. D.; Goel A. K.; Jindal S. K.; Sinha N. K. Birth of a femalekid from in vitro matured and fertilized caprine oocytes. Indian J.Anim. Sci. 78(6): 680–85; 2008.

Kobayashi K.; Yamashitu S.; Hushi H. Influence of epidermal growthfactors and transforming growth factors on in vitro maturation ofcumulus enclosed bovine oocytes in defined medium. J. Reprod.Fertil. 12: 439–446; 1994.

Kono T.; Obata Y.; Yoshimizu T.; Nakahara T.; Carroll J. Epigeneticmodifications during oocyte growth correlates with extended par-thenogenetic development in the mouse. Nat. Genet. 13: 91–94;1996.

Kure-bayashi S.; Miyake M.; Okada K.; Kato S. Successful implanta-tion of in vitro-matured electro-activated oocytes in the pig.Theriogenology 53: 1105–1110; 2000.

Lagutina I.; Lazzari G.; Duchi R.; Galli C. Developmental potential ofbovine androgenetic and parthenogenetic embryos: a comparativestudy. Biol. Reprod. 70: 400–405; 2004.

Liu L.; Yang X. Parthenogenetic development and protein patterns ofnewly matured bovine oocytes after chemical activation. Mol.Reprod. Dev. 49: 298–307; 1998.

Loi P.; Ledda S.; Fulka J.; Cappai P.; Moor R. M. Development ofparthenogenetic and cloned ovine embryos: effect of activationprotocols. Biol. Reprod. 58: 1177–1187; 1998.

Ongeri E. M.; Bormann C. L.; Butler R. E.; Melican D.; Gavin W. G.;Echelard Y.; Krisher R. L.; Behboodi E. Development of goatembryos after in vitro fertilization and parthenogenetic activationby different methods. Theriogenology 55: 1933–1945; 2001.

Pankaj R. S.; Satisha K. B.; Venkatesh V.; Gupta R.; Kumar K.; Das B.C.; Majumdar A. C.; Bag S.; Ranjan R. Effect of different acti-vation protocol on generation of parthenogenetic embryos incaprine. Indian J. Anim. Sci. 82(11): 1323–1326; 2012.

Presicce G. A.; Yang X. Nuclear dynamics of parthenogenesis ofbovine oocytes matured in vitro for 20 and 40 h and activatedwith combined ethanol and cycloheximide treatment. Mol.Reprod. Dev. 37: 61–68; 1994a.

Presicce G. A.; Yang X. Parthenogenetic development of bovine oo-cytes matured in vitro for 24 h and activated by ethanol andcycloheximide. Mol. Reprod. Dev. 38: 380–385; 1994b.

Ranjan R.; Singh R. K.; Yasotha T.; Kumar Manish.; Puri Gopal.;Kumar Kuldeep.; Singh Renu.; Bhure Sanjeev.; Malakar D.;Bhanja S. K.; Sarkar M.; Das B. C.; Bag Sadhan. Effect of actinpolymerization inhibitor during oocyte maturation on partheno-genetic embryo development and their ploidy in Capra hircus.Biochem. Genet. 2013. (in press).

490 RANJAN ET AL.

Sathisha K. B. Studies on development of parthenogenetic embryos ofdifferent ploidy in buffalo. M.V.Sc. Thesis Submitted to IVRI,Deemed University, Izatnagar, Bareilly-243 122 (UP), India.2009.

Venkatesh V. Development of parthenogenetic stem cells of buffaloand their characterization with pluripotent markers. M.V.Sc.Thesis Submitted to IVRI, Deemed University, Izatnagar,Bareilly-243 122 (UP), India. 2009.

Wani G. M. Plasma progesterone concentrations during oestrus cyclein goats. Indian J. Anim. Reprod. 10(1): 44–46; 1989.

Yi Y. J.; Park C. S. Parthenogenetic development of porcine oocytestreated by ethanol, cycloheximide, cytochalasin B and 6-dimethylaminopurine. Anim. Reprod. Sci. 86: 297–304; 2004.

Young L. E.; Schnieke A. E.; McCreath K. J.; Wieckowski S.Conservation of IGF2-H19 and IGF2R imprinting in sheep; effectof somatic cell nuclear transfer.Mech. Dev. 120: 1433–1442; 2003.


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