Small Ruminant Research 97 (2011) 94–100

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Small Ruminant Research

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Short- and long-term effects of low estrogenic subterranean clover onewe reproductive performance

V. Pace ∗, G. Contò, F. Carfì, A. Chiariotti, G. CatilloCRA-PCM Animal Production Research Centre, Via Salaria 31, 00015 Monterotondo, Rome, Italy

a r t i c l e i n f o

Article history:Received 21 September 2010Received in revised form 24 February 2011Accepted 24 February 2011

Keywords:PhytoestrogensSubterranean cloverReproductionEwes

a b s t r a c t

This study examined the short- and long-term effects on the reproductive performanceof sheep by subjecting them to varieties of subterranean clover (Trifolium subterraneum),containing a low content of total phytoestrogens and formononetin. In the first trial, 30 ewelambs of the Sarda breed (initial mean live weight 16.4 ± 1.3 kg) were divided into threegroups of 10 animals each and maintained separately on pastures for 60 d, with alfalfa(Medicago sativa L.), oats (Avena sativa), and subterranean clover with a phytoestrogencontent ranging from 10.21 mg/g on a dry matter basis (DM) at the onset, to 0.90 mg/gDM at the end of the trial, respectively. All three pastures were integrated to make themisoproteic and isoenergetic. At the end of the grazing period, the animals were pooled intoone group and fed the same phytoestrogen-free diet. No differences were recorded betweenthe groups with regard to the development of the reproductive system as monitored bytransvaginal ultrasonography, fertility, fecundity, breeding performance of the ewes, orwith regard to the birth weight and conformation of the lambs. Puberty was attained earlierby the animals fed on subterranean clover, while the lambs also weighed more. In thesecond trial, 24 ewe lambs of the Comisana breed (initial mean live weight 25.0 ± 3.4 kg)were divided into two groups of 12 animals each. All animals were fed ad libitum for a20 months period with fresh-cut or dried subterranean clover (phytoestrogen content of0.88 and 0.81 mg/g DM, respectively); or a control non-estrogenic diet, based on fresh-cutor dried Italian ryegrass (Lolium multiflorum Lam.). The two diets were integrated so to beisoenergetic and isoproteic. No differences were recorded between the groups with regardto the attainment of puberty, although the animals fed clover were heavier at puberty. Thedevelopment of the reproductive system, fertility, fecundity and breeding performancein both lambings, the interval between lambings, and body weight and conformation ofthe lambs did not show differences. In both trials, neither non-degraded phytoestrogensnor equol (metabolite of formononetin) were recorded in the blood of the ewes. The onlymetabolite present was p-ethylphenol. All animals fed subterranean clover-based diets,

showed a significantly higher ADG, compared to those fed the control diets. The resultsof this study indicate that the levels of total phytoestrogens and formononetin in the newvarieties of subterranean clover do not negatively affect the reproduction of sheep andappear to improve the gro

∗ Corresponding author. Tel.: +39 0690090226; fax: +39 069061541.E-mail address: vilma.pace@entecra.it (V. Pace).

0921-4488/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.smallrumres.2011.02.011

wth rate of the animals.© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Researchers have noted disturbances in ruminantreproduction, emanating from the ingestion of sub-terranean clover (SC), containing high quantities ofphytoestrogens (PEs) and isoflavones that have an estro-

inant Re

V. Pace et al. / Small Rum

genic activity (Adams, 1977, 1990, 1995; Firth et al., 1977;Kallela et al., 1984).

Formononetin and its ruminal metabolite, equol, causethe most negative effects of all the PEs, with equol hav-ing the greater estrogenic activity (Shutt and Braden, 1968;Shutt et al., 1970; Lloyd Davies and Hill, 1989). Rumenmicro-organisms extensively transform the other estro-genic isoflavones present in the SC, principally genisteinand biochanin-A, into a p-ethylphenol – a substance withno hormonal activity (Lundh et al., 1990). However, recentin vitro studies have shown that p-ethylphenol can stim-ulate the production of prostaglandin-F2� in the corpusluteum in a way similar to estrogen (Woclawek-Potockaet al., 2006).

Due to the close structural similarity of the PEs(phytoestrogens) to mammalian steroid hormones, someresearchers have speculated that in animals, PEs couldhave anabolic effects, similar to those of the sex hor-mones (Trenkle and Borroughs, 1978; Greiner et al., 2001;Moorby et al., 2004). Furthermore, the chemical structureof the isoflavones and their metabolites confer the capac-ity on these substances to bind themselves to estrogen� and � receptors (Kuiper et al., 1998) where they dis-play both estrogenic and anti-estrogenic activity (Cassidyand Faughnan, 2000). Recent studies in different animalspecies have highlighted the numerous biological effectsof PEs, such as, e.g., higher plasma growth hormone(GH) and insulin-like growth factor (IGF-1) concentrations(Moorby et al., 2004); the stimulation of GH productionin the central nervous system (CNS) (Misztal et al., 2007);increased blood calcium levels and bone density, and animproved feed conversion index (Ni et al., 2007). The in vivoeffects of PEs have been the subject of a series of testsin laboratory animals (mice) and monogastrics (rabbits),in which, in addition to their normal diet, these animalswere fed a lyophilized alcoholic extract of SC (subter-ranean clover). In all the tests, the animals that receivedPEs demonstrated an average daily gain significantly higherthan the controls (Pace et al., 1994; Pace and Settineri,1996).

Studies involving Friesian calves (both bull and cas-trates) and fed a diet based on fresh SC (total PEs 0.92%on a DM basis), have highlighted the positive effect on thegrowth in bull calves. The castrated animals exhibited apositive effect regarding ADG only in the first month, beforeshowing an abrupt decline during the subsequent month(Settineri et al., 2002). Furthermore, prepubertal Friesianheifers pasture fed SC (total PE of 0.17% on a DM basis),exhibited an ADG that was significantly higher than that ofthe control group – with no effect on the onset of puberty(Pace et al., 2001). Currently, new varieties of subterraneanclover (SC) have been selected for a reduced content notonly in formononetin, but also in phytoestrogens (PEs),with values that do not exceed 0.5% to 1% DM. The aimof the present study was to verify whether these levelsof PEs, and in particular of formononetin (administered

over varying periods of time), are associated with a reduc-tion in the fertility of ewes, alterations in the reproductiveorgans, have negative effects on the course of gestationand lambing, or cause morphologic modifications to theoffspring.

search 97 (2011) 94–100 95

2. Materials and methods

2.1. Reagents and materials

Standards of the isoflavones genistein (4′ ,5,7-trihydroxyisoflavone),formononetin (7-hydroxy-4′-methoxyisoflavone), daidzein(4′ ,7-dihydroxyisoflavone), and biochanin-A (5,7-dihydroxy-4′-methoxyisoflavone) were obtained from Sigma–Aldrich (Milan, Italy).P-ethylphenol, equol (4′ ,7-isoflavandiol), methanol, and water, the latterboth being HPLC grade, while the other organic solvents of analyticalgrade, also supplied by Sigma–Aldrich (Milan, Italy). Standard stocksolutions of each compound were prepared by dissolving the standardsin a suitable quantity of methanol. Working solutions containing eachof the 4 compounds were prepared by dilution of the standard stocksolutions, and stored at −20 ◦C. Glucuronidase–sulfatase of Helix pomatiawas purchased from Sigma–Aldrich (Milan, Italy). The cartridge C18employed for solid phase extraction of the plasma was obtained fromWaters (Milford, MA, USA) and the 0.45 �m PTFE filters acquired fromMillipore (Billerica, MA, USA). Plasma progesterone (P4) assays wereperformed using a DSL-3900 P4 RIA Kit (DSL, Webster, TX, USA), andvalues were recorded in a gamma-counter (Cobra II, Packard A, CamberraCompany (USA).

2.2. Animals and experimental design

2.2.1. Trial 1In trial 1, 30 ewe lambs of the Sarda breed, with an initial mean live

weight of 16.4 ± 1.3 kg, were subdivided into three groups and fed isopro-teic and isoenergetic diets for 2 months, as follows: Group A, grazing onalfalfa + 300 g/d maize grain + 200 g/d sunflower meal; Group B, grazingoats + 250 g/d maize grain + 250 g/d sunflower meal; and Group C, grazingsubterranean clover (SC) + 300 g/d maize grain + 200 g/d sunflower meal.Three cultivars of SC (Karridale, Northam and Nuba, purchased from S.A.G.,Olbia, Italy), selected on the basis of their low PEs and formononetin con-tent, were sown in autumn. When the plants reached a height of 15 cm,samples of the whole plant and of the leaves were collected every 2 weeks.At the end of the first grazing period, the lambs were reunited into onegroup, and two fertile rams introduced. The group was then fed the samenon-estrogenic standard diet for 8 months. During the grazing trial andthe three following weeks, blood was sampled weekly by jugular punctureat three specific times on the same day (08h00, 13h00, and 18h00). Theplasma samples were then used to determine the concentrations of PEsand metabolites and progesterone (P4) concentration. Ewe lambs wereconsidered to have attained puberty when the P4 plasma levels werehigher than 1.0 ng/ml.

A month after the introduction of the rams, tests were initiated toconfirm pregnancies and the status of the reproductive system by meansof transvaginal ultrasonography. The lambing date was also used to verifythe breeding times. Gestation length was assumed to be 150 d.

The course of the lambing and the litter number, birth weight, andconformation of the lambs born were recorded.

2.2.2. Trial 2In trial 2, 24 ewe lambs of the Comisana breed with an initial mean

live weight of 25.0 ± 3.4 kg were subdivided into two groups and fed fora 20 month period in a sheepfold on isoproteic and isoenergetic diets, asfollows: Group A: SC (cultivars Antas, Limbara and Trikkala, purchasedfrom S.A.G., Olbia, Italy) ad libitum + 300 g/d maize grain + 250 g/d sun-flower meal; and Group B: (control, PE-free); Italian ryegrass (IR) adlibitum + 250 g/d maize grain + 350 g/d sunflower meal. During the periodApril–June (spring), the forage was cut daily and provided fresh. Subse-quently, the same forage was fed as hay. During the trial, weekly bloodsamples were taken from the jugular vein for the determination of theplasma PE and their metabolite concentrations and, up until the attain-ment of puberty, for the level of P4. The ewes were considered to haveattained puberty when plasma P4 levels were higher than 1.0 ng/ml.

Two fertile rams were then introduced into each of the two groups,and after a month, transvaginal ultrasonography was used on a monthly

basis, to confirm pregnancies and evaluate the condition of the repro-ductive system. The lambing date was used to verify the breeding timeswhile the gestation length was assumed to be 150 d. Data were recordedregarding the course of the births, the number of offspring, birth weightand conformation of the lambs born. For verification of the long-termeffects of PEs on fertility and reproduction, the ewes were mated again

96 V. Pace et al. / Small Ruminant Research 97 (2011) 94–100

Table 1Chemical composition of forages used in trial 1 (A, alfalfa; B, oats; C, subterranean clover) and trial 2 (IR, Italian ryegrass; SC, subterranean clover).

Trial 1 Trial 2

A B C IR SC IR hay SC hay

DM % 17.6 15.8 11.9 25.1 17.8 91.2 89.1Ash (%/DM) 11.3 6.9 13.3 8.5 10.4 7.3 9.7CP (%/DM) 16.8 10.8 17.0 11.8 17.6 9.8 13.4EE (%/DM) 2.8 1.9 2.5 1.0 1.4 2.0 2.3NDF (%/DM) 33.4 36.2 28.7 44.3 24.8 64.3 44.3ADF (%/DM) 28.6 30.6 21.6 29.2 19.8 39.6 31.2PE (mg/g) n.d. n.d. 5.56 n.d. 0.88 n.d. 0.81

C d detergB rraneans (DM 873 and 250

asl

2

ufAaiateitgdTItw1esciuTgtma(aatct(

2

ca

2

Y

w((

P, crude protein; EE, ether extract; NDF, neutral detergent fibre; ADF, aci(oats) 250 g/d maize grain and 250 g/d sunflower meal; Group C (subte

upplement of diets was Group IR (Italian ryegrass) 250 g/d of maize grain7.2% on DM basis); Group SC (subterranean clover) 300 g/d maize grain

pproximately 3 months after the first lambing. Similar transvaginal ultra-onography, number of offspring, birth weight and conformation of theambs born, as described for the first lambing, were recorded.

.3. Sample preparation and analyses

The phytoestrogen content of the forages (Table 1) was determinedsing the Lloyd Davies and Hill (1989) method, modified as follows: all theorage samples were manually collected in different parts of the meadow.pproximately 3 kg of the fresh plant material was collected for each for-ge and a sub-sample of 1 kg was homogenized using a Waring blender,n ethyl alcohol. The alcohol extract was dried, redissolved in water,nd extracted three times in separator funnels with petroleum ether,o remove pigments and lipids. The mixed aqueous phases were againxtracted three times with ethylic ether, and the extract dried, redissolvedn methanol, and analyzed with the aid of a HPLC. The analysis of PEs andheir metabolites was determined using a Perkin Elmer liquid chromato-raph, with a model 250 controller pump connected to a photodiode-arrayetector, model 200 (Perkin Elmer Instruments LLC, Shelton, CT, USA).he column was a reversed phase C18 Inertsil ODS-2 (250 mm × 4.6 mm.D.; 5 �m; Varian Inc., Turin, Italy), operating at room temperature, withhe mobile phase being water (A) and methanol (B). The gradient profileas 0–7 min from 25 to 50% B (gradient slope: 1), 7–22 min from 50 to

00% B (gradient slope: 1.5), and then from 22 to 27 min, 100% B (gradi-nt slope: 0). The flow rate being 1 ml/min and the detection wavelengthet at 254 nm. Standard chemical analysis of forages, according to Asso-iation of Official Agricultural Chemists (AOAC, 1980) methods, is shownn Table 1. Plasma stored at −20 ◦C was analyzed for P4 concentration,sing a DSL-3900 P4 RIA Kit obtained from DSL Laboratories (Webster,X, USA), validated for use on sheep plasma and the values recorded by aamma-counter (Cobra II, Packard A Canberra Company, USA). The sensi-ivity of the assay was 0.03 ng/ml. The dosage of the plasma PEs and their

etabolites was determined as follows: 3 ml of plasma mixed with 0.5 mlcetate buffer (0.2 M, pH = 4) and filtered through C18 RP-SPE columnsWaters, Milford, MA, USA). The eluate was dried and incubated for 16 ht 37 ◦C after mixing with 0.5 ml of a freshly prepared mixture of 10 mlcetate buffer (0.2 M, pH = 5) and 50 �l glucuronidase/sulfatase (H. poma-ia, Sigma). The hydrolyzed samples (total of unconjugated and hydrolysedonjugated PEs and their metabolites) were filtered and centrifuged prioro HPLC injection, according to the procedure used for subterranean cloverSC) extracts.

.4. Statistical analysis

All data were analyzed using the GLM (General Linear Model) pro-edure with SAS software (SAS, 2006). The different parameters weressessed by adopting appropriate statistical models, as described below.

.4.1. Trial 1

The model for the analyses of p-ethylphenol was

ijk = m + Pi + Hj + (P × H)ij + eijk (1)

here m is the overall mean, Pi the fixed effect for months of pasturagei = 1, 2; beginning and end), Hj the fixed effect for hour of samplingj = 1,. . . 3: hours 08h00, 13h00, and 18h00), and eijk the random effect

ent fibre; PE, phytoestrogens (DM 90.33%, CP 37.2% on DM basis); Groupclover) 300 g/d maize grain and 200 g/d of sunflower meal. In trial 2 the.51%, CP 9.31% on DM basis) and 350 g/d sunflower meal (DM 90.33%, CPg/d sunflower meal.

of error. For the weights, increases, and different ages examined up to theattainment of puberty of the sheep, the model was

Yij = m + Gi + eij (2)

where m is the overall mean, Gi the fixed effect for food group (i = 1,. . . 3:alfalfa, ryegrass, and clover), and eij the random effect of error.

The model used for lamb birth weight was

Yijk = m + Gi + Sj + (G × S)ij + eijk (3)

where m is the overall mean, Gi the fixed effect for food group (i = 1,. . . 3:alfalfa, ryegrass, and clover), Sj the fixed effect for sex (j = 1, 2: male andfemale), and eijk the random effect of error.

2.4.2. Trial 2Body weights, age at puberty, and the interval between lambings

were analyzed using model 2 of the first trial in which the fixed effectGi was comprised only of two levels (i = 1, 2: clover and ryegrass). Thebirth weight of the lambs in the two consecutive lambings was analyzedusing model 3, where the fixed effect Gi was comprised of only two lev-els (i = 1, 2: clover and ryegrass). The significance of the differences of theestimated averages was evaluated using the Student’s t-test. Reproduc-tive parameters of fertility, fecundity, and prolificacy were estimated forthe ewes used in both trials.

3. Results

3.1. Trial 1

Fig. 1 illustrates the PE (phytoestrogen) content of thesubterranean clover (SC) used within the timeframe ofthe trial (April–May; spring) and the formonetin:genisteinratio. No detectable quantities of estrogenic isoflavoneswere detected in the alfalfa and the oats (Table 1). Dueto the particularly arid season, the availability of pastureswas limited to a period of 2 months. The quantity of genis-tein and biochanin-A was higher in the initial vegetativestate of the plants and then decreased rapidly with time.Formononetin followed the same trend, although alwaysmaintaining very low levels. PEs were present mainly inthe leaf extracts in a concentration ratio of 5:1, relative tothe other plant components. This result partially explainsthe decreasing trend of the PEs quantities. Leaf weightpercentage diminished with time in the growing stems(Spanu et al., 1993). Moreover, PEs extraction from the

vegetable tissue became more difficult following their pro-gressive dehydration resulting from their growth and theapproaching dry season. The total amount of SC-detectablePEs ranged from 10.2 mg/g DM at the onset, to 0.9 mg/gDM at the end of the trial. Formononetin and its metabo-

V. Pace et al. / Small Ruminant Research 97 (2011) 94–100 97

0

0.1

0.2

0.3

0.4

1 2 3 4 5 6

F/G

ra�

o

Sampling �mes (weeks)

-1.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

1 2 3 4 5 6

PE m

g/g

Genistein

Biochanin

Formonone�n

netin/genistein ratio (F/G) of subterranean clover in trial 1.

Table 2Concentration of ewe plasma p-ethylphenol (�g/ml), in the first and sec-ond months of subterranean clover (SC) grazing in trial 1.

08h00 13h00 18h00 Total

First month 6.41Aa 11.13Ba 2.67Ca 20.21a

Second month 3.04ABb 5.24Ab 2.08Ba 10.36b

Sampling �mes (weeks)

Fig. 1. Trend in phytoestrogen content and formono

lite equol were not detected at any time in the plasmasamples. The only metabolite detected in the ewe plasmawas p-ethylphenol, and its levels were directly related tothe genistein and biochanin-A levels of the SC. The highestquantity of this metabolite was recorded after about 30 dof grazing, and the values decreased significantly duringthe second month (Table 2). The maximum value for thep-ethylphenol content was observed at 13h00. The plasmaP4 concentrations (Fig. 2) did not display any significant

differences between lambs from the three groups duringthe entire pasture period. At the end of this period, noneof the lambs had reached puberty (plasma P4 levels above1 ng/ml), but did so afterwards – during the period of col-lective feeding without PEs (Table 3). Group C (SC + 300 g/d

Fig. 2. Trend of progesterone (P4) in the plasma of the sheep during grazing (0–6B grazed on oats; Group C grazed on clover.

Table 3Body weight (kg), average daily gain (ADG, g/d) and reproduction parameters of e

Groups N Initial liveweight

ADG in thegrazing period

Weight atpuberty

ADG in the postgrazing period

A 10 23.3 141.1b 36.5 47.71a

B 10 22.8 146.1b 34.9 31.91b

C 10 22.4 175.3a 35.4 30.64b

RMSE 1.793 23.364 3.013 0.010

a,bValues within a row with different superscripts differ significantly (P < 0.05) inFertility rate (number of lambing ewes/ewes mated).Prolificacy rate (number of lambs born/100 lambings).Fecundity rate (number of lambs born/ewes mated).RMSE: root mean square error.

c From the beginning of grazing.

RMSE 1.873

a,bP < 0.05 in the column; A,BP < 0.05 in the row.RMSE: root mean square error.

maize grain + 200 g/d sunflower meal) was on average ear-lier by about 20 d, compared to Group A (alfalfa + 300 g/dmaize grain + 200 g/d sunflower meal) and by 40 d, withrespect to Group B (oat + 250 g/d maize grain + 250 g/d sun-

0 d) and post-grazing periods in trial 1. Group A grazed on alfalfa; Group

we lambs and weights at birth of offspring in trial 1.

Onset ofpuberty (d)c

Fertility%

Prolificacy%

Fecundity%

Male(kg)

Female(kg)

182ab 90 100 90 3.62 3.08201a 70 114 80 3.03 3.10162b 90 111 100 2.77 3.06

30.677 0.153 0.199

the column.

98 V. Pace et al. / Small Ruminant Research 97 (2011) 94–100

0

0.2

0.4

0.6

0.8

1PE

mg/

g

Genistein Biochanin Formonone�n

0.01

0.02

0.03

0.04

F/G

ra�

o

netin/g

flap9b

tftwd

twfbudcd

3

s

TBa

a

FPFR

1 2 3 4 5

Sampling �mes (weeks)

Fig. 3. Trend in phytoestrogen content and formono

ower meal) regarding puberty. Two months after allnimals had reached puberty, the numbers of confirmedregnancies were 9/10 in Group A, 7/10 in Group B, and/10 in Group C, which corresponded to the lambing num-ers (Table 3).

The ADG of the lambs fed subterranean clover duringhe grazing period was significantly higher than for thoseed alfalfa and oats. The ADG suddenly decreased whenhe animals were transferred to the pooled feeding regime,ithout PEs. Thus, at puberty, there were no significantifferences in body weight between the three groups.

In Table 3 the reproductive parameters and weights ofhe lambs at birth are set out. No significant differencesere again recorded between the groups with regard to

ertility, prolificacy, and fecundity, or with regard to theirth weight or number of lambs born. The transvaginalltrasonography did not reveal any abnormalities in theevelopment of the reproductive system, or during theourse of the pregnancies, and no difficulties were detecteduring lambing. No abnormal lambs were born.

.2. Trial 2

The varieties of subterranean clover (SC) used in theecond trial had a lower PE content – particularly that

able 4ody weight (kg) of ewes, average daily gain (ADG, g/d) age at puberty and at firstt the first (1) and second (2) lambing in trial 2.

Groups N Initial live weight Weight at puberty ADG (150 d)

SC 12 29.2 45.0a 104.9a

IR 12 27.4 39.0b 77.5b

RMSE 5.880 3.714 0.025

N Lambing Fertility % Prolifica

SC 12 1 100 117IR 12 100 125RMSE

SC 12 2 92 127IR 10c 80 125RMSE

,bValues within a row with different superscripts differ significantly (P < 0.05) inertility rate (number of lambing ewes/ewes mated).rolificacy rate (number of lambs born/100 lambings).ecundity rate (number of lambs born/ewes mated).MSE: root mean square error.c In the period following the first lambing, two sheep from the control group d

1 2 3 4 5

Sampling �mes (weeks)

enistein ratio (F/G) of subterranean clover in trial 2.

of formononetin – compared to the varieties used inthe first trial (Fig. 3). Genistein was present in higherquantities relative to the other components, and the for-mononetin/genistein ratio was lower, compared to the firsttrial (Fig. 3). The average content of total PEs in the hay(0.81 mg/g DM) was very similar to that present in the freshherbage (0.88 mg/g DM), at the time of cutting and dry-ing. No detectable quantities of estrogenic isoflavones werefound in the Italian ryegrass (Table 1).

Similar to the findings of the earlier trial, neither for-mononetin nor equol were found in the plasma of theanimals fed SC, and the only metabolite present was p-ethylphenol. The mean concentrations being 2.02 �g/mlduring the period of feeding with fresh herbage and1.94 �g/ml during the period of feeding with hay. No differ-ences were recorded between the two groups with regardto concentrations of plasma P4. Further, the presence of PEsin the diet did not affect the onset of puberty and the ageat first lambing (Table 4), or the interval between the twolambings. The ADG of the two groups, however, was appre-

ciably different, as was the weight at puberty. The animalsfed SC showed a higher growth trend than those of the con-trol group over the entire observation period, including theonset of the trial and the onset of puberty – with both freshand dry clover.

lambing (d), reproduction parameters, number and weight (kg) of lambs

Onset of puberty Age at first lambing Inter-lambing period

233 383 241231 381 241

14.487 14.487 11.457

cy % Fecundity % Male (kg) Female (kg)

117 4.98 4.62125 4.68 4.07

0.2000 0.2087

127 5.02 3.94110 3.95 4.30

0.3429 0.2851

the column.

ied from accidental causes.

inant Re

V. Pace et al. / Small Rum

The presence of PEs in the diet also did not influencefertility, fecundity, and prolificacy (Table 4), either overthe short- or long-term. The transvaginal ultrasonogra-phy revealed no anomalies or morphological changes ofthe reproductive organs and no abortions. Furthermore, nodifficulties were recorded during birth, with no abnormallambs being born.

4. Discussion

The administration of SC to pre-pubertal lambs, con-taining moderate or low levels of total PEs and very lowquantities of formononetin, appeared to have had no effecton their reproductive capabilities – regardless of the con-ditions or length of the trial. However it did significantlyincrease body growth. In the first experiment, carried outon recently weaned animals grazing in a field of legumescontaining PEs (C), grasses (B), and legumes without PEs(A). There were only indications of a significant precocityor early puberty in Group C, compared to Group B regardingpuberty-without repercussions on the overall reproduc-tive performance of the animals or the characteristics ofthe lambs born. The absence of detectable quantities offormononetin and equol in the plasma of the lambs andthe presence of p-ethylphenol as the only metabolite, indi-cated that appreciable levels of genistein and biochanin-Ain the forages did not significantly affect the reproductiveparameters – if the ratio formononetin:genistein remained<0.5.

Dickinson et al. (1988) demonstrated, rumen microor-ganisms to extensively transform genistein and biochanin-A into p-ethylphenol, a substance with no hormonal activ-ity. However, according to data published by Woclawek-Potocka et al. (2005a,b, 2006, 2008), p-ethylphenol seemsto have an effect similar to that of equol in the stimula-tion of prostaglandin secretion from cells of the corpusluteum, and the bovine endometrium. In a series of in vitroand in vivo trials on cows fed with soybean, a variationin absorption and metabolism of PEs was noted, depend-ing on the physiological and hormonal state of the animals.It was also reported that a reduction in fertility could beattributed to the action of the active metabolites equol andp-ethylphenol on the secretion of prostaglandin and on theinhibition of the secretion of LH-stimulated P4. Therefore,the negative effects on the fertility of cows would at leastin part result from the reduction in the secretion of P4,induced by prostaglandin. In the present trial, although p-ethylphenol was always found in the plasma of the sheepfed SC, there was no evidence of any negative effect onfertility that could be linked to the biological mechanismassumed in previous research on cows. However, it shouldbe emphasized that the duration of the grazing feeding trialwas only 2 months and that the potential effects on fer-tility may not have been detected. In this regard, Adams(1995) differentiated a temporary infertility, which ceasedbetween 4 and 6 weeks after the termination of the inges-

tion of PEs – from a permanent infertility that resulted froma prolonged ingestion of even low quantities of estrogenicisoflavones. The latter was characterized by irreversiblehistological and functional changes in the reproductive sys-tem.

search 97 (2011) 94–100 99

The second trial, involving continuous ingestion of SCwith a low and constant content of total PEs over the entireexperimental period, confirmed the absence of effects. Inthe short-term, the onset of ovarian cyclic activity, normalonset of the estrus, progress of pregnancies, and reproduc-tive parameters were highlighted by the absence of effects,even following a longer period of treatment. After approx-imately 20 months from the onset of the second trial, nodifferences were recorded regarding fertility, fecundity, orprolificacy relative to the control group. Furthermore, noembryonic deaths, births of malformed lambs, or subclin-ical forms of reduced fertility were recorded (Millingtonet al., 1964; Adams, 1977, 1990, 1995; Kallela et al., 1984).However, it should be stressed that the sample size wassmall in both trials, and effects on fertility cannot be com-pletely excluded if a larger sample size was to be used.

In both trials, the PEs had a significant effect on thegrowth of the animals. In trial 1, the ADG of the lambsin Group C (SC + 300 g/d maize grain + 200 g/d sunflowermeal) was significantly greater than that of the other twogroups, over the entire grazing period – even though thediets were isoenergetic and isoproteic. The clover andalfalfa in particular, had a similar protein content, bothin quantity and nutritional quality, with the main differ-ence between the two diets being the presence of PEs.In this regard, it should be noted that Andersen et al.(2009a,b), using a liquid chromatography (LC)–mass spec-trometry (MS/MS) technique, detected a very low quantity,even lower than 1 ppm, estrogenic isoflavones in the alfalfasilage, the maize silage, the barley, the sugar beet pulpand the rapeseed cake. These values are approximately100–1000 times lower than those present in the varietiesof subterranean clover used in this trial and their effectson the growth of the ruminants have not yet been inves-tigated. The abrupt decrease in growth found in Group Cafter the end of the grazing period and the transition to anon-estrogenic diet, therefore, appears to indicate an effectattributable to these substances.

Trial 2 yielded a similar result, although the PE contentof the clover used was much lower than that of trial 1, with aformononetin:genistein ratio being less than 0.1. This find-ing agrees with the results of a trial on ram lambs in whichthe animals fed SC had an ADG approximately 32% higherthan the control group (Pace et al., 2006). It could thereforebe inferred that the quantity of substances that promotegrowth is lower than the quantity of substances that effectreproduction.

Several previous results seem to support this hypoth-esis. Misztal et al. (2007) reported a stimulation of GHsecretion at the CNS level in sheep treated with genistein.Ni et al. (2007) however reported increased expression ofthe gene GH-R and IGF-IR, despite a higher level of hematiccalcium and a higher feed conversion ratio in hens fed dietscontaining low levels of daidzen. Han et al. (2008) demon-strated a positive effect of daidzen (at a level of 5 mg/L)in the synthesis of volatile fatty acids (VFA) in the rumen

of goats, and the disappearance of any effect when thelevel of daidzen was increased to 20 mg/L. This last findingconfirms earlier observations in mice fed a standard diet,supplemented with a purified alcoholic extract of PEs at adosage of 100 mg/kg or 1 g/kg of feed – in which the high-

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st body weight gain was obtained with the lowest level ofdded PEs (Pace et al., 1996). Such results seem to be relatedo the capacity of the estrogenic isoflavones to show estro-enic or anti-estrogenic activity, depending on the dosageCassidy and Faughnan, 2000).

. Conclusions

A low phytoestrogen (PE) content and, above all, a lowormononetin content, such as those present in the newarieties of subterranean clover (SC), do not appear tonterfere negatively on the reproductive processes of ewes,nd in fact favour body growth. Among the estrogenicsoflavones, genistein appears to be mainly responsible forhis effect, and diets that contain this substance could besed as natural growth promoters for ruminants. How-ver, research into the mechanism by which PEs affectnimal reproduction and body growth still yield contra-ictory results, that call for more extensive research.

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