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Animal Reproduction Science 142 (2013) 10– 18

Contents lists available at ScienceDirect

Animal Reproduction Science

journa l h om epa ge: www.elsev ier .com/ locate /an i reprosc i

tudies on enhancing embryo quantity and quality bymmunization against inhibin in repeatedly superovulatedolstein heifers and the associated endocrine mechanisms

.P. Liua, X.B. Maob, Y.M. Weib, J.N. Yuc, H. Li c, R.A.Chena, Z.D. Shic,∗

Guangdong Enterprise Key Laboratory of Biotechnology R&D of Veterinary Biologics, Zhaoqing DaHuaNong Biology Medicine Co., LTD.,haoqing 526238, ChinaCollege of Animal Science, Guangxi University, Nanning 530000, ChinaLaboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing10014, China

a r t i c l e i n f o

rticle history:eceived 31 December 2012eceived in revised form 6 August 2013ccepted 8 August 2013vailable online 19 August 2013

eywords:nhibinmmunizationmbryo productionormonesolstein heifers

a b s t r a c t

To investigate the feasibility of improving embryo production in cattle by immunizationagainst inhibin, both in vivo and in vitro experiments were conducted. In two experimentsconducted in two autumns, 14 animals aged 14 months were immunized with a recombi-nant inhibin � subunit protein antigen for four times at monthly intervals, with another14 animals of the same age served as the controls. Starting from the second immuniza-tion, all the heifers received standard superovulation treatment for three sessions, onesession per month, each starting 10 days after every antigen administration. Immunizationagainst inhibin increased number of transferable embryos (P < 0.05), and high quality GradeA embryos (P < 0.01) in each superovulation. Blood concentrations of FSH, estradiol, activin,and also ratio of activin to follistatin concentrations were greater in inhibin immunizedthan in control animals during the period of superovulatory FSH administration and ani-mal estrous expression. Heifers immunized with inhibin also had greater concentrations ofprogesterone in the later diestrus period. In the second experiment, the inclusion of anti-inhibin antibody in oocyte IVM medium increased oocyte maturation rate and cleavage ratefollowing IVF (P < 0.05). These results demonstrated that inhibition of the adverse effects ofinhibin on ovarian follicular development and oocyte maturation improved embryo yield,

in both quantity and quality, following superovulation. These results also demonstrate thatactive immunization against inhibin, in conjunction with the conventional superovulationprotocol, can constitute a new technique for consistent improvement of bovine embryoproduction in vivo; while passive immunization with anti-inhibin antibody can improve

in vitr

embryo production

. Introduction

Multiple ovulation and embryo transfer (MOET) tech-

ique has long been applied to dairy and beef cattlereeding programs (Gearheart et al., 1989; McGuirk, 1989;uo et al., 2009). MOET hastens the generation intervals

∗ Corresponding author. Tel.: +86 2584390956; fax: +86 2584390345.E-mail addresses: zdshi@jaas.ac.cn, zdshi01@qq.com (Z.D. Shi).

378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.anireprosci.2013.08.005

o.© 2013 Elsevier B.V. All rights reserved.

and improves genetic progress, especially when it is com-bined with marker-assisted selection techniques (Luo et al.,2009). However, a conventional MOET operation consistsof several periods of hormone treatments, yet can produceonly five to six transferable embryos on average, whilerequiring skillful expertise and resulting greater labor

charges. MOET is thus regarded as an expensive and inef-ficient procedure (Hasler, 2003). The needs to reduce thecost of embryo transfer (ET) and to further boost ET appli-cation require the development of more efficient embryo

oduction

Y.P. Liu et al. / Animal Repr

producing superovulation techniques (Hasler, 2003). Thisnecessity has been addressed by laboratories that havemodified the conventional superovulation protocol thatrequired treatment with bovine somatotropin (bST) toenhance support to the development of ovarian folli-cles (Gong et al., 1993, 1996; Moreira et al., 2002). Thelarge amount of inhibin secreted by multiple developingfollicles (Kaneko et al., 1992; Taya et al., 1996), how-ever, might negatively impact follicular development, ovafunction and quality during follicular atresia and selec-tion (Austin et al., 2001; Jimenez-Krassel et al., 2003;Lu et al., 2009). Immunoneutralization of inhibin couldinhibit the detrimental effects of this hormone to matur-ing oocyte and also the resulting embryos, as evident inthe consistent improvement in embryo yield and qual-ity following conventional superovulation protocols (Meiet al., 2009; Li et al., 2009, 2011). Nevertheless, in previ-ous studies immunization against inhibin occurred onlyby a single superovulation procedure (Mantovani et al.,1997; Takedomi et al., 2005; Mei et al., 2009; Li et al.,2009). The resulting single improvement of embryo yieldstill does not warrant the long term increase in embryoproduction efficiency as wished by the ET industry. Thepresent study investigated the feasibility of consistentlyimproving embryo production in cattle by immuniza-tion against inhibin, in heifers superovulated for threeconsecutive times at monthly intervals. The changes inreproductive hormones during and after superovulationprocedure were monitored. The anti-inhibin antibody wasutilized for in vitro maturation and fertilization of cattleoocytes to illustrate the endocrine mechanisms underlyingthe improvement of embryo quality following immuniza-tion against inhibin (Mei et al., 2009; Li et al., 2009).

2. Materials and methods

2.1. Immunogen

The porcine inhibin � subunit fusion protein harbor-ing an N-terminal (His)6 tag was expressed using pRSET-Avector (Invitrogen) in E. coli. BL21 (DE3), and purified aspreviously described (Li et al., 2009). This recombinant pro-tein was purified with Ni-NTA resin (Qiagen) to greaterthan 90% purity. The purified inhibin � subunit was homog-enized with mineral oil adjuvant and added to a mixture ofwater and Grade 10 Injection white oil (Hangzhou Refinery,Hangzhou, China) at the ratio of 4:6 (v:v), to reach final con-centration of 1 or 0.5 mg/ml for use as immunogen in theexperiment. The control immunogen was prepared withphysiological saline homogenized with mineral oil adju-vant. The above experimental and control immunogenswere employed to produce effective immune reaction andembryo yields, as described previously (Mei et al., 2009; Liet al., 2009).

2.2. Animals and treatments

The experiment was conducted in two autumns in 2years on one dairy farm. In each autumn, 14 Holsteinheifers aged 14 months were divided equally into the Con-trol group (n = 7) that was immunized with the control

Science 142 (2013) 10– 18 11

immunogen, and the Treated group (n = 7) that was immu-nized with inhibin antigen. On days 1, 28, 56 and 88 of the110-days experimental course, the Treated group receivedi.m. injections of 1 ml inhibin immunogen containing 1 mgof the recombinant inhibin protein. Control heifers wereeach similarly treated with 1 ml control immunogen at thesame time period as treatment applications to the Treatedgroup. In the first autumn experiment, blood samples werealso collected for antibody titration and hormone concen-tration measurements. These, together with immunizationand superovulation arrangements, are depicted in detail inFig. 1.

During the course of the experiment, all the animalswere raised in a hygienic environment with sufficient andcomfortable free range area were fed with concentratefeed and corn silage according to nutritional requirement,and had free access to drinking water. The experimentwas approved by the Research Committee of JiangsuAcademy of Agricultural Sciences under the guidance ofthe Regulations for the Administration of Affairs Concern-ing Experimental Animals (Decree No.2 of the State Scienceand Technology Commission on November 14, 1988).

2.3. Superovulation, insemination and embryo flushing

On the sixth day after each immunization except theprimary immunization, which is designated as day 1 ofeach superovulation procedure, each heifer received a pro-gesterone releasing intravaginal devices (PRID; ShanghaiInstitute of Planned Parenthood Research) containing 1.9 gof progesterone. Four days later, designated as day 4 ofeach superovulation procedure, each animal received ani.m. injection of FSH (Folltropin-V, Bioniche Animal HealthCanada Inc.), twice daily, for four consecutive days indiminishing dosage of 70 mg, 50 mg, 30 mg and 15 mg. Onthe third day of FSH administration, all heifers receivedtwo i.m. injections, 8 h apart, of 0.4 mg cloprostenolprostaglandin (PG) (Shanghai Institute of Planned Parent-hood Research). The PRID was withdrawn immediatelyafter the second PG administration. After completion ofFSH administrations, the heifers were all monitored forestrous expression. On the ninth day of each superovula-tion procedure (the day after the last FSH injection), theheifers were artificially inseminated twice, 12 h apart. Onthe seventh day following artificial insemination (day 16 ofeach superovulation procedure), embryos were collectedfollowing non-surgical flushing, counted and examined oftheir quality according to criteria in the Manual of Inter-national Society of Embryo Transfer (Stringfellow & Seidel,1998). The embryos were categorized to Grades A, B, C ordegenerated, on the basis of morphology, color, uniformityand size of blastomere; and of thickness and intactness ofzona pellusida. The total number of Grades A and B embryoswere counted as transferables, whereas the degenerativeembryos and non-fertilized ova as non-transferables.

2.4. Blood sampling

All animals in the Control (n = 7) and Treated (n = 7)group in the first autumn experiment were used forblood sample collections. The samples were collected by

12 Y.P. Liu et al. / Animal Reproduction Science 142 (2013) 10– 18

Fig. 1. Experimental scheme of the immunization, superovulation procedures in both autumns, and also blood collection for antibody titer measurementconducted only in the first autumn experiment. Wide arrows indicated administration of inhibin immunogen. Small arrows and droplets indicate bloods (SO) sta1 of bloodF

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ampling for measurement of anti-inhibin antibody titers. Superovulation5 days later. During the second superovulation procedure, a second set

ig. 3.

enipuncture from tail vein into a tube containing 100 IUeparin, on days 1, 15, 28, 44, 58 and 88 of the experi-ent to measure anti-inhibin antibody titer. To measure

ormone concentrations during and after superovulationrocedure, a second set of blood samples was also collecteduring and after the second round of superovulation proce-ures. Starting from onset of FSH treatment, samples wereollected once daily for 4 days, and then collected twelveimes every other day to cover a period longer than a nor-

al estrous cycle. All blood samples were centrifuged at000 g for 20 min within 2 h of collection, to separate thelasma, and stored at −20 ◦C until being analyzed.

.5. Measurements of antibody titer and hormoneoncentrations in blood

Standard ELISA method was utilized to measure anti-nhibin antibody titer in plasma. The recombinant inhibin

subunit fusion protein was used to coat the 96-wellicro-titer plate (5 �g/well in 100 �l). After blocking with

% skim milk and washing, a duplicate 100 �l plasmaample, diluted 1:800 with 1% skimmed milk, was addedo each well. The samples were incubated for the bind-ng of anti-inhibin antibody to the coated inhibin fusionrotein. The bound antibody was further labeled byddition of horse-radish peroxydase-labeled rabbit-anti-ovine antibody (Santa Cruz Biotechnology, CA, USA).etection of binding was initiated by the addition ofhromogen tetramethyl benzidine (Sigma Chemical Co., Stouis, USA) solution containing 0.03% H2O2, and was termi-ated as appropriate with the addition of 2% H2SO4. Opticalbsorbance at 450 nm representing anti-inhibin antibodyiter for both Treated and Control heifers was measured.o overcome treatment bias in assay results, plasma sam-les from all heifers that were collected from the sameollection event were measured on the same plate.

Concentrations of plasma estradiol and progesteroneere measured using medical diagnosis RIA kits (Beijingorthern Biotechnology Institute, Beijing, China). To mini-ize possible interference that might be caused by steroid

inding proteins, plasma samples were pre-heated in 70 ◦Cater bath for 30 min to denature the bound proteins and

o release the hormone. Then samples in duplicate werenalyzed according to the assay protocols for estradiol or

rted 6 days after each booster immunization, and embryos were collected samples were collected for assaying hormone concentrations shown in

progesterone supplied by the kit provider. Assay sensitiv-ity, measuring range and intra-assay coefficient of variationfor RIA of estradiol were 1 pg/ml, 1–160 pg/ml and lessthan 10%, respectively; and 0.2 ng/ml, 0.5–10 ng/ml andless than 10% for progesterone assay, respectively. All thesamples were measured in one single assay for either estra-diol or progesterone.

Plasma concentrations of FSH, activin and follistatinwere determined with sandwich ELISA kits (R&D Systems®

China Co., Ltd., Shanghai). These methods were vali-dated for measuring bovine plasma samples by showinga parallelism between inhibition curves of serially dilutedsamples and standard concentrations. All the final sampleresults were the mean of calculated readings of duplicateELISA OD450 values. The assay sensitivities were 0.1 IU/ml,0.1 ng/ml and 10 pg/ml for FSH, activin and follistatin,respectively. The intra- and inter-assay coefficients of vari-ations were less than 10%, for all three ELISA assays.

2.6. Oocyte in vitro maturation, in vitro fertilization andembryo culture

Cow ovaries were obtained from a local slaughterhouse and transported to the laboratory in sterile phys-iological saline at 25∼35 ◦C within 3 h. Oocytes in theform of cumulus–oocyte complexes were aspirated fromfollicles of 2–6 mm in diameter, in wash medium (TCM-199 + 5.0 mM NaHCO3 + 5 mM Hepes + 2% fetal calf serum),by a 10-ml syringe with a 12G injection needle. Afterwashing twice in the wash medium, oocytes were in vitromatured (IVM) in 10-mm culture dish under a humidifiedatmosphere containing 5% CO2 in air at 38.5 ◦C. The baseIVM medium was TCM-199 (Sigma, Missouri, USA), sup-plemented with 26.2 mM NaHCO3 (Sigma), 5 mM Hepes(Sigma), 5% fetal calf serum, 2% bovine follicular fluid (col-lected without regard to the stage of the reproductivecycle) and 100 ng/ml porcine FSH (Sigma). The treatmentwas conducted by adding 0, 50, 100 or 200 �g/ml anti-inhibin antibody (Li et al., 2011) into the base IVM medium.After maturation for 20–22 h, all of the cumulus oophrus

complexes were transferred into 0.1% hyauronidaseenriched Dulbecco’s phosphate buffered saline for 2 min.The surrounding cumulus cells were then mechanicallyremoved by repeated pipetting with a micro-needle under

oduction Science 142 (2013) 10– 18 13

Fig. 2. Anti-inhibin antibody titer in inhibin immunized (�, n = 7) andControl (�, n = 7) group heifers (results were obtained in the first autumn

Y.P. Liu et al. / Animal Repr

stereomicroscope. The matured oocytes that developed toMII stages, showing the first polar body in the perivitellinespace, were counted for the calculation of oocyte matura-tion rate.

For IVF, frozen semen straw (0.25 ml per straw) wasthawed for 10 s in a 37 ◦C water bath, and the spermatozoawere suspended in 1.5 ml improved Tyrode’s medium for30 min. The superstratum phase was aspirated for washingby centrifugation (500 × g for 20 min). Spermatozoa pel-let was re-suspended in improved Tyrode’s medium at aconcentration of 2.0 × 106 sperm/ml for subsequent fertil-ization. The matured MII stage oocytes were washed twice,then transferred into a 30-�l droplet of sperm contain-ing improved Tyrode’s medium (10–15 oocytes per drop),left incubated for 24 h in a humidified atmosphere contain-ing 5% CO2 at 38.5 ◦C. The presumptive zygotes were thenwashed for three times with embryo culture medium toremove excess sperms (TCM-199 supplemented with 3%fetal calf serum) and co-cultured with cow granulosa cellin humidified air containing 5% CO2 at 38.5 ◦C. Every 48 h,half of the cultured medium was changed. Embryo devel-opment was regularly monitored. The number of oocytescleaved was counted after co-culturing for 48 h, and dur-ing days 6 and 8 of culture, numbers of blastocysts wererecorded.

2.7. Statistical analyses

The data of anti-inhibin antibody titers, FSH, estradiol,progesterone, activin, follistatin concentrations and theratio of activin to follistatin concentration were analyzedusing the Mixed model by SAS statistics software (Version9.0, SAS Institute, Cary, NC, USA). The treatment group andthe sampling time designated as fixed factors and heiferidentity as the random factor. The means were comparedby Tukey–Kramer method. In the analyses and comparisonsof the numbers of follicles, embryos and ova, transferableembryos and transferable rate, one-way analysis of vari-ance were adopted. Before ANOVA was performed, theembryo data of two years were combined for each super-ovulation session, to achieve sufficient sample size for eachtreatment group. Data of oocyte maturation rate, cleavagerate and blastocyst rate were compared using the t-testmethod for data of percentage values.

3. Results

3.1. Anti-inhibin antibody titer

Antibody titers were represented by the optical den-sity (OD) value measured at 450 nm photospectrum wavelength in the ELISA test. Throughout the experiment, ODvalues were barely detected in the plasma samples of theControl heifers, as values of 0.05 or less were consideredto be the non-specific binding of the assay (Fig. 2). In thesamples of inhibin immunized Treated heifers, anti-inhibintiters increased after the primary immunization, and in

15 days were greater (P < 0.01) than the pre-immune non-specific binding levels, and were also greater (P < 0.01) thanthe mean of control heifer samples. As immunizations wererepeated, anti-inhibin antibody titers in the Treated group

experiment). Vertical bars represent standard error of the mean. Asterisksindicate means of the Treated group were significant different from thoseof the controls (**P < 0.01). Arrows indicate inhibin immunonization.

heifers increased further, peaking on day 58, which weremaintained on day 88 when the animals were administeredwith the third booster immunization (Fig. 2).

3.2. Embryo quantity and quality

In the first year experiment, embryo collections wereincomplete in one Control heifer after the first superovu-lation treatment and in two Treated animals after the firstand third superovulation treatments, due to difficulties inembryo flushing in some animals arising from overreactionto anesthesia used, and the development of endometri-tis in others. These problems were not encountered inthe second year experiment. Therefore, embryo collec-tion data of these problem animals in the first year wereexcluded from statistical analysis. When data of each col-lection time were combined in two years, average means(±SEM) of data from the remaining animals were shownin Table 1. Compared with that in the Control group,the number of total embryo/ova collected was marginallygreater in the Treated heifers after the first superovula-tion (9.2 ± 1.1 compared with 7.3 ± 1.2, P > 0.05), but wasgreater after the latter two superovulations (10.6 ± 0.9compared with 8.0 ± 1.0 and 10.5 ± 1.1 compared with7.9 ± 1.1, both P < 0.05). From all of three superovulationprocedures, the yield of transferable embryos was greater(P > 0.05) in the Treated heifers (approximately six to sevenembryos) than in the Control group (between three to fourembryos; Table 1). The enhancement of embryo yield wasalso reflected by greater numbers (P < 0.01) of Grade Aembryos, approximately six embryos in the Treated groupin all three superovulations, compared with about twoin the Control group (Table 1). The numbers of Grade B,

degenerated embryos and unfertilized ova, were all simi-lar between the two treatment groups from each of threesuperovulation procedures (Table 1).

14 Y.P. Liu et al. / Animal Reproduction

Tab

le

1M

ean

s

(±S.

E.M

.)

of

vari

ous

grad

es

of

embr

yos

or

un

fert

iliz

ed

ova

reco

vere

d

from

hei

fers

in

the

inh

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and

con

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ps.

a,b

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tion

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grad

es

of

embr

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ova

Perc

enta

ges

of

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grad

es

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ova

to

tota

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bryo

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a

Gra

de

A

Gra

de

B

Deg

ener

ated

Un

fert

iliz

ed

Tota

l em

bryo

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Tran

sfer

able

Gra

de

A

Gra

de

B

Deg

ener

ated

Un

fert

iliz

ed

Tran

sfer

able

Firs

tTr

eate

d

12

5.3

±

1.0c

0.4

±

0.1a

1.1

±

0.3a

2.1

±

0.5a

9.2

±

1.1a

5.9

±

1.0b

57.4

±

4.6c

4.7

±

2.2b

12.4

±

3.5c

23.3

±

5.1a

64.3

±

3.7c

Con

trol

13

1.7

±

0.2a

1.4

±

0.4a

2.4

±

0.6a

1.7

±

0.6a

7.3

±

1.2a

3.1

±

0.4a

23.5

±

4.3a

19.6

±

4.8a

33.3

±

5.4a

23.5

±

6.4a

43.1

±

4.7a

Seco

nd

Trea

ted

14

6.1

±

0.6c

0.6

±

0.2a

2.0

±

0.4a

1.9

±

0.4a

10.6

±

0.9b

6.7

±

0.7c

57.8

±

3.7c

6.9

±

1.9c

16.8

± 2.

6a18

.9

±

3.8a

64.3

±

4.0a

Con

trol

14

2.1

±

0.3a

1.5

±

0.3a

2.3

±

0.5a

2.1

±

0.7a

8.0

±

1.0a

3.6

±

0.4a

32.2

±

6.4a

18.4

±

3.6a

27.8

± 4.

6a21

.0

±

5.4a

51.2

±

5.2a

Thir

dTr

eate

d

12

6.3

±

0.5c

0.7

±

0.2a

1.7

±

0.6a

1.9

±

0.6a

10.5

±

1.1b

6.9

±

0.6c

60.3

±

4.7c

6.2

±

1.7b

16.4

± 3.

5b17

.8

±

5.5a

65.8

±

5.0a

Con

trol

14

2.4

±

0.4a

1.4

±

0.3a

2.3

±

0.5a

1.7

±

0.6a

7.9

±

1.1a

3.9

±

0.5a

26.1

±

4.2a

18.0

±

6.9a

28.8

±

4.8a

21.6

±

6.1a

49.6

±

6.2a

aW

ith

in

each

sup

erov

ula

tion

sess

ion

, dat

a

wer

e

the

com

bin

ed

resu

lts

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two

autu

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s

in

2

year

s.b

The

per

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tage

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each

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gory

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ova

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e

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of

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coll

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in

each

sup

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pro

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ure

, dat

a

wit

hou

t

the

sam

e

lett

er

in

the

sam

e

colu

mn

sup

ersc

rip

t

dif

fer

(a–b

:

P

<

0.05

;

a–c:

P

<

0.01

).

Science 142 (2013) 10– 18

On the percentage basis, Treated heifers yielded agreater percentage, approximately 65% in all three super-ovulations, of transferrable embryos to total number ofembryos and unfertilized ova, as compared with 43–50% inthe Control animals. There was a difference (P < 0.01) for thefirst superovulation (Table 1). The Treated animals also hada greater (P < 0.01) proportions of the high quality Grade Aembryos (approximately 60%), but fewer (P < 0.05) Grade Band degenerated embryos, than those in the Control group(Table 1).

3.3. Plasma hormone concentrations

The hormone concentrations measured in the secondset of blood samples collected during and after the secondsuperovulation procedure are shown in Fig. 3. Follow-ing the commencement of FSH administration, plasmaFSH concentrations steadily increased in both groups ofheifers, but the increase was more rapid and greater(P < 0.01) in the Treated than in the Control group fromthe 3rd day of FSH administration at the time whenthe peak, or post-ovulation FSH peak was occurring 2days after the last or 4th day of FSH administration, andafter the estrous expression and ovulation (Fig. 2a). Amuch smaller second peak occurred in the Treated, butnot in Control heifers post FSH administration, on days22–24, which is after 18–20 days in an analogous estrouscycle.

Plasma estradiol concentrations displayed a similarchanging pattern as that of FSH, although the peak con-centrations occurred on the fourth or last day of FSHadministration in both groups of animals (Fig. 2b), 2 daysearlier than the FSH peak. Again, the concentrations weregreater (P < 0.01) in the Treated than Control animals,during superovulatory FSH administration stage and thelater diestrus period. After cloprostenol administrationand PRID withdrawal, plasma progesterone concentra-tions decreased to very low concentrations on the dayof estrous expression and peak estradiol concentration,which then steadily increased and reached the peak 10days later. Progesterone concentrations then decreased tothe nadir on day 21 post-estrus (Fig. 3c). Again, inhibin-immunized heifers had greater (P < 0.01) progesteroneconcentrations surrounding the peak than did the Controlanimals.

Changes in plasma follistatin concentrations were sim-ilar to those of estradiol, exhibiting the similar riseassociated with FSH treatment and the decline afterwards(Fig. 3d). The peak follistatin concentrations also occurredat the same time as that of estradiol, which was slightlyless in Treated than in Control animals. Plasma activin con-centrations peaked on the third day of FSH administration(Fig. 3e), which was earlier than the peak of estradiol andfollistatin. Also, throughout the entire 25 day samplingperiod which covered the follicular phase and the subse-quent luteal phase, Treated heifers had greater (P < 0.01)activin concentrations than the Controls. This made the

activin:follistatin concentration ratio greater in Treatedthan in Control groups of animals (Fig. 3f), and the differ-ences were significant between the two groups of animalsduring the follicular phase around FSH administration.

Y.P. Liu et al. / Animal Reproduction Science 142 (2013) 10– 18 15

Fig. 3. Plasma concentrations of FSH (a), estradiol (b), progesterone (c), and activin (d), follistatin (e) and activin to follistatin concentration ratio (f) inwere obnt from

and FSH

inhibin immunized (�, n = 7) and Control (�, n = 7) group heifers (results

error of the mean. Asterisks indicate mean of Treated group were differefour arrows in (a) indicate implantation of PRID, injection of cloprostenol

3.4. Effects of anti-inhibin antibody on oocyte IVM andIVF embryo development

An addition of anti-inhibin antibody at dosages of50 �g/ml or 100 �g/ml into the maturation mediumincreased the oocyte maturation rate from 72.7% withoutuse of antibody, to 76.5% (P > 0.05) and 80.3% (P < 0.05),respectively (Table 2). However, when dosage was fur-ther increased to 200 �g/ml, maturation rate decreased to69.6%, which is similar to the rate at the control dosage.After IVF, the cleavage rates exhibited a changing pat-tern similar to that of maturation rate between the dosageamounts (Table 2), increasing from 87.5% without use ofantibody, to 92.0% (P > 0.05) and 96.7% (P < 0.05), respec-tively, upon addition of the antibody at dosages of 50 �g/mlor 100 �g/ml. The addition of the anti-inhibin antibody hadno effect on blastocyst rate, or the percentage of maturedoocytes developing to blastocyst stage (P > 0.05). Uponintroduction of the anti-inhibin antibody at the greatestconcentration (200 �g/ml), both cleavage and blastocystrates decreased to concentrations less than that of the Con-trol rates obtained without use of anti-inhibin antibody(P < 0.01; Table 2).

4. Discussion

To improve long-term embryo production in bovine,this study observed the effects of immunization against

tained in the first autumn experiment). Vertical bars represent standard that of Control Group (*P < 0.05; **P < 0.01). Thick dash line, X mark and, respectively.

inhibin on embryo production in heifers undergoing super-ovulation procedures for three consecutive sessions atmonthly intervals. In accordance with previous reportsof improving embryo production (Li et al., 2009; Meiet al., 2009), inhibin immuno-neutralization in this studyconsistently improved the total number of embryos andunfertilized ova, the yield of transferable embryos, and theyield and proportion of high quality Grade A embryos. Inthe present study the combination of inhibin immuniza-tion with a conventional superovulation protocol improvedlong term embryo production in cattle, with enhancementof both embryo quantity and quality.

Apart from one study that did not observe an increasein embryo yield and quality in inhibin immunized super-ovulated heifers (Mantovani et al., 1997), several others allreported increased total ovulation numbers, as indirectlyrepresented by the numbers of harvested embryos andunfertilized ova (Takedomi et al., 2005; Li et al., 2009; Meiet al., 2009). Likewise, in the present study there was also anincrease in the total numbers of embryos and ova as a resultof inhibin immunization. The increment enlarged frombeing marginal to significant after repeated immunizationsand superovulations. This resulted in an approximately70–90% greater yield in the number of transferable embryo

in the Treated than in the Control animals. In addition, thenumber of Grade A embryos was equivalent to 60% of thetotal number of embryos and ova collected, twice as highas in the control group. These results obtained following

16 Y.P. Liu et al. / Animal Reproduction Science 142 (2013) 10– 18

Table 2Oocyte maturation and IVF embryo development following the addition of anti-inhibin antibody in the oocyte maturation medium.

Dosage (�g/ml) Oocytes cultured Maturation rate (%)a Oocytes fertilized Cleavage rate (%)b Blastocyst rate (%)c

0 77 72.7 (56)a 56 87.5 (49)a 39.3 (22)a

50 81 76.5 (62)ab 62 92.0 (57)ab 40.3 (25)a

100 76 80.3 (61)b 61 96.7 (59)a 41.0 (25)a

200 69 69.6 (48)a 48 61.8 (42)c 25.0 (17)c

Numbers in the parenthesis are the actual numbers of oocytes, embryos or blastocysts. Within a column, values without a common superscript differ (a–b:P < 0.05, a–c, b–c: P < 0.01).

a dy out

ytes for

for fert

ir2ecpbTutApdo

eptnthvlfiteKmcStorpibelhadlodoso

Maturation rate = number of oocytes matured or showing first poly bob Cleavage rate = number of cleaved zygotes out of total number of oocc Blastocyst rate = number of blastocysts out of total number of oocytes

mmunization against inhibin are all consistent with thoseeported from previous studies (Li et al., 2009; Mei et al.,009). The overall embryo yield less than four transferablembryos in the Control heifers in the present study was lessompared with the yield of six to eight embryos obtained inrevious studies (Takedomi et al., 2005; Mei et al., 2009),ut comparable to those reported by Gong et al. (1993).his phenomenon could be due to the young age of heiferssed in the present study, or to do with plane of nutri-ion fed to the animals (Boland et al., 2001; Gong, 2002).nother possible cause for the lesser embryo yield in theresent study could be the monthly superovulation proce-ure, which might alter the ovarian follicular developmentrder and affect the developing follicle cohort number.

Similar to the results from previous studies (Medant al., 2003, 2004), immunization against inhibin elevatedlasma concentrations of FSH. Although the preovula-ory surge that should occur on the day of estrus wasot detected due to blood sample collection schedule,he post-ovulatory FSH peak was greater in immunizedeifers than that in the control animals. A second ele-ation of plasma FSH concentration occurring 16 daysater, at the time of progesterone concentration decrease,urther illustrates the stimulation of FSH secretion bymmunoneutralization of inhibin. As was demonstratedhat immunoneutralizing inhibin bioactivity stimulatesstradiol secretion by cultured granulosa cells (Jimenez-rassel et al., 2003), plasma estradiol concentrations wereuch greater in inhibin-immunized heifers than in the

ontrols prior to and shortly after the estrous expression.ecretion of considerable amount of estradiol after ovula-ion might indicate the presence of intact follicles in thevaries, a phenomenon previously seen in superovulatedats (Szołtys et al., 1994). Nevertheless, the production ofrogesterone in luteal phase was accordingly greater in

nhibin-immunized heifers in the present study, as hadeen observed in other studies (Medan et al., 2004; Meit al., 2009; Li et al., 2011), which indicated more corporautea and, therefore, more ovulated follicles. Another twoormones associated with ovarian follicle development,ctivin and follistatin, also showed variation in secretionuring and after superovulation procedure. While the fol-

istatin concentrations changed in a pattern similar to thatf estradiol, the activin concentrations reached the peak 1

ay before that of follistatin, a phenomenon similar to thatbserved in buffalo cows (Li et al., 2011). This difference inecretion may also reflect the difference in the follicle originf the two hormones – small sized follicles secrete activin,

of total number of oocytes × 100.fertilization × 100.ilization × 100.

while large follicles secrete follistatin (Knight & Glister,2001). Plasma activin remained at greater concentrationsin inhibin-immunized animals, possibly because these ani-mals consistently possessed numerous small sized follicles,as reported previously (Medan et al., 2004).

A major finding of this study was the increase inGrade A or high quality embryos following immuniza-tion against inhibin. This phenomenon has been previouslyreported in the improvement of female embryos followinginsemination of sex-sorted semen in inhibin-immunized,superovulated Holstein heifers (Li et al., 2009). In this previ-ous study, improvement of embryo quality was attributedto greater estradiol and progesterone secretions followingneutralizing inhibin bioactivity. Apart from such enhanc-ing roles by ovarian steroids of embryo developmentalcompetence (Mann and Lamming, 1999), in vitro results ofthe present study suggest direct neutralization of inhibinbioactivity by the addition of anti-inhibin antibody inoocyte maturation medium enhanced oocyte maturationrate, thus improving embryo quality. It is, therefore, rea-sonable to expect that for oocytes treated with anti-inhibinantibody, the increase in oocyte quality would bring abouta similar increase in embryo developmental competence,as indicated by increased cleavage rate, if not improvementin blastocyst rate. These results of improvement in embryoquality were consistent with those obtained from anti-inhibin antibody-treated buffalo oocytes (Li et al., 2011).Oocytes used for in vitro maturation in this and otherstudies were in the form of cumulus-oocyte complexes.The cumulus cells surrounding the oocytes were capableof synthesizing and secreting local auto/paracrine regula-tory factors such as inhibin, activin and follistatin (Knightand Glister, 2001, 2006). These factors are all retainedin the culture medium during oocyte IVM, and throughcell microvilli, could influence the development and func-tion of the cumulus cells, as well as the ovum itself andthe subsequent IVF embryo. For example, cumulus–oocytecomplexes in IVM could secrete up to >100 ng/ml of bothactivin and follistatin, and possibly inhibin as well, in thematuration medium (Silva and Knight, 1998; Silva et al.,1999). Though oocyte that contained follistatin stimulatesembryo developmental competence (Patel et al., 2007; Leeet al., 2009; VandeVoort et al., 2009), follistatin and inhibin� subunit secreted into the maturation medium impedes,

while activin enhances oocyte maturation and embryodevelopment (Silva and Knight, 1998; Silva et al. 1999;Yoshioka et al., 1998; Izadyar et al., 1998a,b,c). It has beenfurther demonstrated that while activin A promotes oocyte

oduction

Y.P. Liu et al. / Animal Repr

maturation and embryo developmental competence until9- to 16-cell stage of embryo development, follistatinattenuates these processes (Yoshioka et al., 1998). Mostimportantly, the activin to follistatin ratio, which deter-mines the presence of free activin that is capable of bindingto its receptors, was shown to increase during oocyte mat-uration (Izadyar et al., 1998a,b,c), and might determineoocyte maturation quality and embryo developmentalcompetence (Silva and Knight, 1998). Therefore, immu-nization against follistatin and/or inhibin would providemore free activin, augment activitin receptor signaling(Chapman et al., 2002; Wiater et al., 2009), enhance oocytematuration and subsequently embryo development (Liet al., 2011).

The above proposed mechanism derived from in vitrostudies may be applied to illustrate the improvement ofembryo quality following immunization against inhibin.In the in vivo condition, plasma inhibin concentrationswere elevated due to the formation of multiple large folli-cles following superovulatory FSH administration (Kanekoet al., 1992). Greater concentrations of secreted inhibin mayexert detrimental effects on the oocyte function withindeveloping follicles, and negatively affect the quality ofthe resulting embryo in the control heifers. However, sucheffect is diminished in inhibin immunized heifers. As forfollistatin, plasma concentrations also rose following FSHadministrations in both the control and inhibin immu-nized heifers. Nevertheless, the ratio of activin to follistatinis higher in inhibin immunized heifers, especially duringthe FSH administration period and also days after, whichwere in the stages of final follicular and oocyte matura-tion and early embryo development. Stronger would bethe net promoting impact in inhibin immunized heiferson oocyte maturation and the subsequent early embryodevelopment. This could cause inhibin-immunized heifersto produce greater proportions or yields of high qualityGrade A embryos.

In summary, immuno-neutralization against inhibinin vivo and in vitro diminishes the adverse effects exertedby inhibin and follistaitn on ovarian follicular devel-opment, oocyte maturation and embryo development.This immuno-neutralization contributes to enhancedembryo quality and quantity. In the present study, activeimmunization against inhibin in conjunction with the con-ventional superovulation protocol was also found to be atechnique for long term improvement of embryo produc-tion in cattle in vivo. Passive immunization, however, mayserve as a novel method to improve embryo productionin vitro.

Acknowledgements

The authors want to thank Drs Shujin Li, Wenli Yu,and Mr. Long Chen of Beijing AnBo Embryo Biotech Cen-ter for providing the animals and operational assistances;and Dr. Qinyou Liu of Institute of Animal Reproduction atGuangxi University for the conduction of oocyte IVM/IVF

experiments. This research was supported by the NationalResearch and Development Grant (2011BAD19B02-6) andJiangsu Agriculture Science and Technology Inovation Func(CX(11)4074).

Science 142 (2013) 10– 18 17

References

Austin, E.J., Mihm, M., Evans, A.C., Knight, P.G., Ireland, J.L., Ireland, J.J.,Roche, J.F., 2001. Alterations in intrafollicular regulatory factors andapoptosis during selection of follicles in the first follicular wave of thebovine estrous cycle. Biol. Reprod. 64 (3), 839–848.

Boland, M.P., Lonergan, P., O’Callaghan, D., 2001. Effect of nutrition onendocrine parameters, ovarian physiology, and oocyte and embryodevelopment. Theriogenology 55 (6), 1323–1340.

Chapman, S.C., Bernard, D.J., Jelen, J., Woodruff, T.K., 2002. Propertiesof inhibin binding to betaglycan. InhBP/p120 and the activin type IIreceptors. Mol. Cell. Endocrinol. 196 (1–2), 79–93.

Gearheart, W.W., Smith, C., Teepker, G., 1989. Multiple ovulation andembryo manipulation in the improvement of beef cattle: relative the-oretical rates of genetic change. J. Anim. Sci. 67, 2863–2871.

Gong, J.G., Bramley, T.A., Wilmut, I., Webb, R., 1993. Effect of recom-binant bovine somatotropin on the superovulatory response topregnant mare serum gonadotropin in heifers. Biol. Reprod. 48 (5),1141–1149.

Gong, J.G., Wilmut, I., Bramley, T.A., Webb, R., 1996. Pretreatmentwith recombinant bovine somatotropin enhances the superovulatoryresponse to FSH in heifers. Theriogenology 45 (3), 611–622.

Gong, J.G., 2002. Influence of metabolic hormones and nutrition on ovarianfollicle development in cattle: practical implications. Domest. Anim.Endocrinol. 23, 229–241.

Hasler, J.F., 2003. The current status and future of commercial embryotransfer in cattle. Anim. Reprod. Sci. 79 (3–4), 245–264.

Izadyar, F., Dijkstra, G., Van Tol, H.T., Van den Eijnden-van Raaij, A.J.,Van den Hurk, R., Colenbrander, B., Bevers, M.M., 1998a. Immuno-histochemical localization and mRNA expression of activin, inhibin,follistatin, and activin receptor in bovine cumulus-oocyte complexesduring in vitro maturation. Mol. Reprod. Dev. 49 (2), 186–195.

Izadyar, F., Hage, W.J., Colenbrander, B., Bevers, M.M., 1998b. The pro-motory effect of growth hormone on the developmental competenceof in vitro matured bovine oocytes is due to improved cytoplasmicmaturation. Mol. Reprod. Dev. 49 (4), 444–453.

Izadyar, F., Zeinstra, E., Bevers, M.M., 1998c. Follicle-stimulating hormoneand growth hormone act differently on nuclear maturation whileboth enhance developmental competence of in vitro matured bovineoocytes. Mol. Reprod. Dev. 51 (3), 339–345.

Jimenez-Krassel, F., Winn, M.E., Burns, D., Ireland, J.L., Ireland, J.J., 2003.Evidence for a negative intrafollicular role for inhibin in regula-tion of estradiol production by granulosa cells. Endocrinology 144,1876–1886.

Kaneko, H., Watanabe, G., Taya, K., Sasamoto, S., 1992. Changes in periph-eral levels of bioactive and immunoreactive inhibin, estradiol-17 beta,progesterone, luteinizing hormone, and follicle-stimulating hormoneassociated with follicular development in cows induced to superovu-late with equine chorionic gonadotropin. Biol. Reprod. 47, 76–82.

Knight, P.G., Glister, C., 2001. Potential local regulatory functions of inhib-ins, activins and follistatin in the ovary. Reproduction 121, 503–512.

Knight, P.G., Glister, C., 2006. TGF-beta superfamily members and ovarianfollicle development. Reproduction 132, 191–206.

Lee, K.-B., Bettegowda, A., Wee, G., Ireland, J.J., Smith, G.W., 2009. Molec-ular determinants of oocyte competence: potential functional rolefor maternal (oocyte-derived) follistatin in promoting bovine earlyembryogenesis. Endocrinology 150, 2463–2471.

Li, C., Zhu, Y.L., Xue, J.H., Zhang, S.L., Ma, Z., Shi, Z.D., 2009. Immunizationagainst inhibin enhances both embryo quantity and quality in Hol-stein heifers after super-ovulation and insemination with sex-sortedsemen. Theriogenology 71, 1011–1017.

Li, D.R., Qin, G.S., Wei, Y.M., Lu, F.H., Huang, Q.S., Jiang, H.S., Shi, D.S., Shi,Z.D., 2011. Immunization against inhibin enhances follicular devel-opment, oocyte maturation and superovulatory response in waterbuffaloes. Reprod. Fert. Dev. 23, 788–797.

Lu, C., Yang, W., Chen, M., Liu, T., Yang, J., Tan, P., Li, L., Hu, X., Fan, C., Hu,Z., Liu, Y., 2009. Inhibin A inhibits follicle-stimulating hormone (FSH)action by suppressing its receptor expression in cultured rat granulosacells. Mol. Cell. Endocrinol. 298, 48–56.

Luo, W., Wang, Y., Zhang, Y., 2009. Simulation study on the efficiencies ofMOET nucleus breeding schemes applying marker assisted selectionin dairy cattle. Sci. China C Life. Sci. 52, 296–306.

McGuirk, B., 1989. The relevance of MOET programmes to developingcountries. Theriogenology 31, 29–40.

Mann, G.E., Lamming, G.E., 1999. The influence of progesterone duringearly pregnancy in cattle. Reprod. Domest. Anim. 34, 269–274.

Mantovani, R., Enright, W.J., Roche, J.F., Boland, M.P., 1997. The effect ofimmunization of heifers against alpha 1-26 inhibin conjugate on thesuperovulatory response to pFSH. Theriogenology 47, 511–519.

1 duction

M

M

M

M

P

S

S

inhibin antagonism in gonadotropes. Mol. Endocrinol. 23 (7),

8 Y.P. Liu et al. / Animal Repro

edan, M.S., Watanabe, G., Sasaki, K., Nagura, Y., Sakaime, H., Fujita, M.,Sharawy, S., Taya, K., 2003. Ovarian and hormonal response of femalegoats to active immunization against inhibin. J. Endocrinol. 177 (2),287–294.

edan, M.S., Akagi, S., Kaneko, H., Watanabe, G., Tsonis, C.G., Taya, K., 2004.Effects of re-immunization of heifers against inhibin on hormonalprofiles and ovulation rate. Reproduction 128, 475–482.

ei, Cheng, Li, M.Y., Zhong, S.K., Lei, Y., Shi, Z.D., 2009. Enhancing embryoyield by immunization against inhibin in superovulated Holsteinheifers. Reprod. Domest. Anim. 44, 735–739.

oreira, F., Badinga, L., Burnley, C., Thatcher, W.W., 2002. Bovine soma-totropin increases embryonic development in superovulated cowsand improves post-transfer pregnancy rates when given to lactatingrecipient cows. Theriogenology 57 (4), 1371–1387.

atel, O.V., Bettegowda, A., Ireland, J.J., Coussens, P.M., Lonergan, P., Smith,G.W., 2007. Functional genomics studies of oocyte competence: evi-dence that reduced transcript abundance for follistatin is associatedwith poor developmental competence of bovine oocytes. Reproduc-tion 133 (1), 95–106.

ilva, C.C., Knight, P.G., 1998. Modulatory actions of activin-A and follis-

tatin on the developmental competence of in vitro-matured bovineoocytes. Biol. Reprod. 58 (2), 558–565.

ilva, C.C., Groome, N.P., Knight, P.G., 1999. Demonstration of the suppres-sive effect of inhibin �-subunit on the developmental competence ofin vitro matured bovine oocytes. J. Reprod. Fert. 115, 381–388.

Science 142 (2013) 10– 18

Stringfellow, D.A., Seidel, S.M., 1998. Manual of the International EmbryoTransfer Society, third ed. International Embryo Transfer Society,Savoy, IL, USA.

Szołtys, M., Galas, J., Jabłonka, A., Tabarowski, Z., 1994. Some morpholog-ical and hormonal aspects of ovulation and superovulation in the rat.J. Endocrinol. 141, 91–100.

Takedomi, T., Kishi, H., Medan, M.S., Aoyagi, Y., Konishi, M., Itoh, T.,Yazawa, S., Watanabe, G., Taya, K., 2005. Active immunization againstinhibin improves superovulatory response to exogenous FSH in cattle.J. Reprod. Dev. 51, 341–346.

Taya, K., Kaneko, H., Takedomi, T., Kishi, H., Watanabe, G., 1996. Role ofinhibin in the regulation of FSH secretion and folliculogenesis in cows.Anim. Reprod. Sci. 42, 563–570.

VandeVoort, C.A., Mtango, N.R., Lee, Y.S., Smith, G.W., Latham, K.E., 2009.Differential effects of follistatin on nonhuman primate oocyte matura-tion and pre-implantation embryo development in vitro. Biol. Reprod.81, 1139–1146.

Wiater, E., Lewis, K.A., Donaldson, C., Vaughan, J., Bilezikjian, L., Vale,W., 2009. Endogenous betaglycan is essential for high-potency

1033–1042.Yoshioka, K., Suzuki, C., Iwamura, S., 1998. Activin A and follistatin regu-

late developmental competence of in vitro-produced bovine embryos.Biol. Reprod. 59 (5), 1017–1022.