Effects of Trifolium alexandrinum phytoestrogens on oestrous behaviour, ovarian activity and reproductive performance of ewes during the non-breeding season

Accepted Manuscript Title: Effects of Trifolium alexandrinum phytoestrogens on oestrous behaviour, ovarian activity and reproductive performance of ewes during the non-breeding season Authors: N.M. Hashem, K.M. El-Azrak, A.N.M. Nour El-Din, S.M. Sallam, T.A. Taha, M.H. Salem PII: DOI: Reference:

S0378-4320(17)31051-5 https://doi.org/10.1016/j.anireprosci.2018.03.007 ANIREP 5785

To appear in:

Animal Reproduction Science

Received date: Revised date: Accepted date:

16-12-2017 26-2-2018 6-3-2018

Please cite this article as: Hashem NM, El-Azrak KM, Nour El-Din ANM, Sallam SM, Taha TA, Salem MH, Effects of Trifolium alexandrinum phytoestrogens on oestrous behaviour, ovarian activity and reproductive performance of ewes during the non-breeding season, Animal Reproduction Science (2010), https://doi.org/10.1016/j.anireprosci.2018.03.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of Trifolium alexandrinum phytoestrogens on oestrous behaviour, ovarian activity and reproductive performance of ewes during the nonbreeding season

N.M. Hashem*, K.M. El-Azrak, A.N.M. Nour El-Din, S.M. Sallam, T.A. Taha, M.H. Salem

Alexandria University, Alexandria 21545, Egypt

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Animal and Fish Production Department, Faculty of Agriculture (El-Shatby),

*Corresponding author: N.M. Hashem, Department of Animal Production, Faculty

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of Agriculture, Alexandria University, Alexandria 21545, Egypt; Tel.:

+2001288719758; Fax: +20 35922780; E-mail: [email protected]

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ABSTRACT

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Phytoestrogens are classified as naturally occurring endocrine disrupting

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chemicals that may affect reproductive performance of farm animals. To investigate the effects of Berseem clover phytoestrogens on reproductive

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performance of seasonal anoestrus ewes, twenty four late pregnant Rahmani ewes were fed either Berseem clover or maize silage (n=12/treatment). Treatment started

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2 months prepartum and continued until oestrous induction (week 8 postpartum), using the CIDR-eCG based protocol, and early pregnancy. Throughout the 2 to 8

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weeks postpartum, oestrous rate and ovarian activity were not affected by treatment. After oestrous induction, ewes in both groups expressed comparable oestrous rates; however feeding Berseem clover extended (P < 0.05) interval to

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oestrus (57.00 compared with 42.54 h) and shortened (P < 0.05) oestrous duration (20.0 compared with 34.90 h). Feeding Berseem clover did not affect follicular activity except the number of medium follicles, which was less (P < 0.05) on day

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of oestrus (Day 0). Feeding maize silage increased (P < 0.05) the total number of follicles and number of small and medium follicles the day before oestrus (Day -1). On Day 0, the greater total number of follicles was due to the greater (P < 0.05) number of medium follicles that was associated with less number of small follicles. Although, the number and diameter of corpora lutea (CLs) were not affected by treatment, serum P4 concentration was greater (P < 0.05) for ewes fed maize silage than for those fed Berseem clover. Fecundity and litter size tended to be greater 1

(about 35%; P = 0.132 and 0.085, respectively) in the maize silage fed ewes. In conclusion, feeding Berseem clover throughout seasonal anoestrus disrupted aspects of behavioural oestrus and there was less luteal P4 synthesis and fecundity of ewes.

Key words: Phytoestrogens; Seasonality; Ewes; Oestrous behaviour; Ovarian

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1.

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activity; Progesterone

Introduction

Breeds of sheep from low (tropical areas: 23ºN-23ºS) and middle (subtropical areas: around 24ºN-34ºN; 24ºS-34ºS) latitudes such as the Mediterranean breeds

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have periods of seasonal anoestrus (lack of oestrous behaviour and ovulation

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activity), although, different proportions of ewes remain oestrous cyclic and can have ovulations (Abu-Zanat, 2005; Gómez-Brunet et al., 2012). In Egypt, the non-

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breeding season of sheep extends from winter to late spring. During this period

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local sheep breeds (Rahmani, Barki and their crossbred) have ovulations (44.4%70%) without associated overt signs of oestrus (“silent oestrus”; Hashem et al.,

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2011; Hashem, 2014). The extent and the length of reproductive seasonality of sheep are regulated by many environmental cues such as photoperiod, nutrition,

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rainfall and the interactions of these factors (Gómez-Brunet et al., 2012). In many Mediterranean basin countries (including Egypt), western and central Asia and South America, Trifolium (T.) alexandrinum,Berseem clover, has been widely

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cultivated in irrigated and rain-fed farming systems as an annual legume roughage crop used in farm animal nutrition (Badr et al., 2008) due to its desirable nutritive value. It, however, has a phytoestrogenic activity because of its considerable

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content of isoflavones. Kolodziejczyk-Czepas et al. (2015) reported that among different T. species (T. fragiferum L., T. hybridum L., T. resupinatum var. majus Boiss., and T. resupinatum var. resupinatum L.), T. alexandrinum had the greatest concentration of total isoflavones (18.97 mg/g dry mass). The main detected isoflavones in many clover species including Berseem clover, red clover (T. pratense) and white clover (T. repens)were genistein, daidzein, biochanin A and formononetein(Steinshamn et al., 2008; Hashem et al., 2016). These phytogenic 2

compoundsare naturally occurring endocrine disrupting agents that may interfere with many reproductive functions of farm animals due to their oestrogen mimicking effects. Earlier studies implicated phytoestrogens in the incidence of many symptoms of reproductive disorders such as reductions in the ovulation and conception rate (Hafez and Hafez, 2000; Ahmed, 2007) and even temporal infertility without apparent visible clinical signs (Adams, 1995).Phytoestrogens could interfere with the oestradiol feedback mechanism to release luteinizing

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hormone (LH) in ewes (Mathieson and Kitts, 1980), and could decrease LH-pulse frequency (Wojik-Gladysz et al., 2005). Infusion of genistein, an isoflavone

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phytoestrogen, into the third ventricle of the brain in ovariectomized ewes during

seasonal anoestrus resulted in a reduction in plasma LH concentrations, frequency of LH pulses and prolactin secretion through actions within the central nervous system (Romanowicz et al., 2004). Phytoestrogens can inhibit aromatase enzyme

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activity in ovarian follicles leading to a decrease in oestradiol (E2) synthesis. In

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addition, phytoestrogens can decrease sensitivity of the pituitary gland to

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gonadotropin releasing hormone (Rosselli et al., 2000). Furthermore, consumption of isoflavones increases the incidence of irregular oestrous cycles and ovulations

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without expression of behavioural oestrus in cattle (Zduńczyk et al., 2005) and ewes (Hashem and Sallam, 2012). Also, feeding Berseem clover induced hormonal

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imbalances in heifers during early pregnancy, resulting in a decrease in conception rate (Hashem et al., 2016). These results when considered together led to

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hypothesis that feeding Berseem clover, a phytoestrogenic roughage rich in isoflavones, wouldaffect the reproductive seasonality of subtropical sheep, particularly the incidence of ovulations without expression of behavioural oestrus.

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This study, therefore, aimed to investigate the effects of feeding Berseem clover on oestrous behaviour, ovarian activity and reproductive performance of subtropical

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Rahmani ewes during the non-breeding season.

2. Materials and methods 2.1. Chemical analyses of experimental diets and isoflavones determination The chemical analyses of the concentrate diet, Berseem clover and maize silage were performed using the methods of AOAC (2000). Furthermore, the 3

concentrations of different isoflavone aglycones including genistein, daidzein, formononetin and biochanin A were determined using standards purchased from Sigma–Aldrich, USA and solvents of high-purity “HPLC” grade (Merck, Darmstadt, Germany) after extraction using the method described by Zgórka (2009). The procedure used for isoflavones determination was conducted using the procedure described by Franke et al. (1994). A HPLC unit connected to a UV-VIS diode-array detector (Dionex-UltiMate 3000) with a reversed-phase C18 (ODS)

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column (250 × 4.5 mm, Dionex-UltiMate 3000) was used. Briefly, a volume of 25

µl sample (extract) or standard was injected at column temperature setting at 35ºC.

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Isoflavones were separated using two mobile phases: phase A 0.1% hydro-acetic

acid (v/v) and phase B 0.1% acetic acid in acetonitrile (v/v) with a total processing time of 20 min. The peaks of samples and standards were detected at 260 nm. The peaks of the separated isoflavones were compared with those of standard

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isoflavones. The concentrations of isoflavones in the samples were calculated in

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terms of µg/g dry matter (DM). The chemical analyses of the concentrate diet,

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Berseem clover and maize silage and their contents of isoflavone aglycones are

2.2. Animals and protocol

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presented in Table1.

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This study was conducted in the Agricultural Experimental Station (31° 20ʹN, 30°E) of the Faculty of Agriculture, Alexandria University, Alexandria, Egypt. The

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experiment was conducted during the period from December to April (winter and spring seasons) representing the non-breeding season, during which the majority

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of females were in lactation as the breeding season starts in June, summer season (Hashem et al., 2011). The Guide for the Care and Use of Agricultural Animals in Research and Teaching, Federation of Animal Science Societies was followed

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during the experimental period (FASS, 2010). Twenty four multiparous Rahmani ewes, aged 3 to 5 years, weighing 41.67±1.83kg with clinically normal and healthy appearance were used. The selected ewes were in the third month of pregnancy and time of parturition was expected during the first week of February (mid non-breeding season). Ewes were equally stratified into two homogenous experimental groups (n=12/group) according to the type of the roughage that was fed. The first group was fed 4

Trifolium alexandrinum (Berseem clover as a phytoestrogenic roughage), while the second group was fed maize silage (a non-phytoestrogenic roughage).The treatment was imposed 2 months preceding parturition (started in the third month of pregnancy) and continued for the next 3.5 months postpartum. The experimental design is depicted in Figure 1. Each experimental group was housed in an open barn with a shelter during the day and in a semi-open barn at the night. The diets offered for both experimental groups were balanced to be isocaloric and

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isonitrogenous, meeting their daily nutritional requirements according to the

physiological status (pregnant or lactating) of the ewes and in accordance to the

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NRC guidelines (1985). The diet was a legume-free concentrate mixture

(containing 390 g/kg maize grains; 300 g/kg cottonseed meal; 275 g/kg wheat bran; 20 g/kg limestone; 10 g/kg NaCl and 5 g/kg mineral premix) and green Berseem clover (clover-group) or maize silage (silage-group) as roughage. The calculated

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mean of total isoflavones intake (four selected isoflavones) provided by each type

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of roughage and the concentrate mixture for each experimental group was

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212076.2µg/ewe/day for the Berseem clover group and 441.2 µg/ewe/day for the

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maize silage group.

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2.3. Detection and induction of oestrus

The signs of behavioural oestrus were detected using “teaser” rams throughout week 2 postpartum until females were synchronized for oestrus at the end of week

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8 postpartum. All ewes were synchronized for oestrus using a controlled internal drug release device (CIDR, Eazi-Breed CIDR, 0.3 g progesterone, Pfizer, New

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Zealand Limited, Auckland) for 8 days with an intramuscular injection of 500 IU of equine chorionic gonadotropin (eCG, CEVA, Syncropart, SANTE Animal, LaBallastière) administered at the time of device removal. To estimate oestrous

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rate, interval to oestrus and duration of oestrus following oestrous induction, the overt signs of oestrus were monitored using “teaser” rams. The “teaser” rams were placed in the pen with ewes twice daily (every 12 h) for 1h following CIDR removal until the termination of overt signs of oestrus. Ewes expressing signs of oestrus were subjected to an assisted natural mating using proven-fertile rams. The interval to oestrus (onset of oestrus) was estimated to be the halfway between the time of CIDR removal and the first positive overt signs of oestrus. The duration of 5

oestrus was estimated to be the halfway between the first acceptance of female to be mounted by the “teaser” ram and the last refusal.

2.4. Monitoring of ovarian activity Ovarian activity was monitored weekly throughout weeks 2 to 8 postpartum using a B-mode transrectal ultrasonography equipped with a 5.0 MHz linear array probe (Pie Medical Equipment B.V., Maastricht, Netherlands). All follicles greater

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than ≥2 mm in each ovary were counted, and then were classified into three

follicular populations: small, ≥2–3 mm; medium, >3–<5 mm; and large (ovulatory)

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follicles, ≥5 mm (Hashem et al., 2015). Furthermore, numbers and diameters of corpora lutea (CLs) were recorded. After induction of the oestrus, number and

classification of the ovarian follicles were conducted 24h after CIDR removal until the time of expression of oestrous behaviour (follicular phase), while numbers and

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diameters of CLs were recorded on day 11 post-mating (luteal phase). Results of

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the ovarian activity during the follicular phase were adjusted in relation to the time

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of initial expression of oestrous behaviour considering day of oestrus as Day 0. Pregnancy diagnoses were conducted by assessing the uterine contents at Day 32

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post-mating.

2.5. Determination of progesterone

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A sample of 10-ml of blood was collected from each ewe into non-heparinized tubes by means of jugular venipuncture during the follicular phase (the day of CIDR removal, 24 and 48h after CIDR removal) and during the luteal phase (Days

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8, 11 and 18 post-mating). Serum was obtained by centrifugation of blood samples at 2000 ×g for 20 minutes and was stored at -20ºC. Concentration of blood serum progesterone (P4) was measured using solid-phase enzyme immunoassay kits

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obtained from Monobind Inc., USA. The lower limit of detection was 0.10 ng/ml serum and the intra- and inter assay CVs were 5.1% and 7.5%, respectively.

2.6. Statistical analysis The MIXED procedure for repeated measurements of SAS (2004) was used. Numbers of total follicles and their classification (according to diameter) as well as 6

numbers of corpora lutea were subjected to square root transformation before tested by MIXED procedure to approximate normal distribution (Howell, 2013). The effects of treatment (Berseem lover and maize silage), time (day of data collection) and the interactions were included in the statistical model as fixed factors. The model was: yijk= µ+Ti+Dj+ (TD)ij+ eijk in which yijk is the observed value of the dependent variable determined from a sample taken from each animal, µ is the overall mean,

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Ti is the fixed effect of the ith treatment, Dj is the fixed effect of the jth day of blood sample collection, (TD)ij is the interaction between treatment and day of ovarian

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scanning or blood sample collection, and eijkis the residual error.

The interval to oestrus and the duration of oestrus were analyzed using the GLM procedure of SAS (2004) using the following model:

yij = µ+ Ti+ eij in which yij is the observed value of the dependent variable, µ is

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the overall mean, Ti is the fixed effect of the ith treatment, and eij is the residual

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error.

Categorical data were expressed as percentages and were analyzed using the

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chi-squared test. Differences among treatment means were tested by Duncan’s

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multiple range test. All results were expressed as least square means (LSM) ±

0.05.

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3. Results

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stander error of the mean (SEM). The statistical significance was accepted at P ≤

Rahmani ewes fed Berseem clover or maize silage failed to express signs of

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oestrus throughout the first 8 week postpartum period. The time to first postpartum ovulation and ovarian activity did not, however, differ between the two groups. The incidence of ovulation and formation of corpus luteum (CL) were not

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associated with overt signs of oestrus (ovulation without expression of behavioural oestrus) throughout the first 8 weeks postpartum (Table 2). After induction of oestrus using CIDR-eCG protocol, ewes fed Berseem clover or maize silage had similaroestrous expression rates (Table 4). Ewes fed Berseem clover had a longer (P < 0.05) interval to oestrus (57.00 h compared with 42.54 h) and shorter (P < 0.05) duration of oestrus (20.0 h compared with 34.90 h). During the follicular phase, ewes fed maize silage had a greater (P < 0.05) total number of 7

follicles at the day before oestrus (Day -1), which was due to the greater (P < 0.05) number of small and medium follicles but not large follicles (Fig. 2). On the day of oestrus (Day 0), there was a greater total number of follicles because of the greater (P < 0.05) number of medium follicles reflecting the improved developmental capacity of the small follicles to medium follicles as the number of small follicles was less (P < 0.05), whereas the number of large follicles was not changed (Figure 2). The follicular activity was not changed by feeding Berseem clover, except

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number of medium follicles which was less (P < 0.05) at the day of oestrus (Fig. 2). Furthermore, the number and diameter of CLs were not affected by type of

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roughage fed to ewes, however, the overall concentration of serum P4 was greater

(P< 0.05) in the maize silage group than in the Berseem clover group (Table 3) due to the incremental increase (P< 0.05) of serum P4 concentrations throughout the days of the luteal phase for ewes fed maize silage (Figure 3). Conception rate at

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Day 32 post-oestrus and lambing rate did not differ (P > 0.05) between both

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groups; however, fecundity and litter size were numerically greater (P = 0.132 and

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0.085, respectively) in ewes fed maize silage than those fed Berseem clover (Table

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4. Discussion

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4).

In the subtropics, the peak of the breeding season in sheep is during the summer season. As a result, most ewes lamb and are in lactation during the early winter to

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late spring, which is classified as the non-breeding season (Hashem et al., 2011; Abu-Zanat, 2005). In the present study, the main objective was to determine whether feeding dietary phytoestrogens for a long term affected reproduction of

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ewes during the non-breeding season. For this purpose, feeding Berseem clover was initiated early in the non-breeding season while the ewes were pregnant. The results of the present study provide evidence that Berseem clover isoflavones had no effect on reproductive performance of ewes to week 8 postpartum. Ewes fed Berseem clover (phytoestrogenic roughage) or maize silage (non-phytoestrogenic roughage) failed to express behavioural oestrus and had the same pattern of ovarian activity (follicular dynamics and time to first ovulation) (Table2). This might 8

suggest implication of other factors in masking the effects of phytoestrogens during the early postpartum period (early lactation). In the presentstudy, ewes lambed in February during the mid non-breeding season. Hashem et al. (2011) reported that, during the non-breeding season, the reduction in oestrous activity during lactation was associated with the month of parturition, with 70.6% of the ewes that lambed in October (late breeding season) expressing oestrous behaviour during lactation, while those that lambed in December failed to express oestrus

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during lactation. Moreover, lactating ewes that lambed during the early breeding

season or towards the end of this period had normal oestrous cycles associated with

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ovarian activities and ovulations. These results together suggest that the transition

from long-day (breeding season) to short-day (non-breeding season) photoperiods is an important factor contributing in the regulation of oestrous behaviour and occurrence of ovulation in subtropical ewes.

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After oestrous induction, feeding Berseem clover, in contrast to maize silage,

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suppressed the developmental capacity of small and medium follicles and,

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therefore, total number of follicles during the days preceding oestrus (Fig. 2). In sheep, the number of recruitable follicles (> 0.2 mm) appears to be dependent on

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follicle stimulating hormone (FSH) concentrations from 24 h preceding induced luteolysis until 60 h after the time of initiation of luteolysis (Driancourt et al.,

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1985). Phytoestrogens suppress basal FSH secretion and also reduce total FSH synthesis (Arispe et al., 2013), which could contribute to the fewer small and

study.

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medium follicles with developmental capacity that were observed in the present

Obvious variations were observed in the present study in oestrous behaviour

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(interval to oestrus and duration of oestrus) between the Berseem clover-fed ewes and those fed maize silage. Considering that the induction of oestrus protocol was imposed in April (toward the end of the out-of breeding season) when the

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photoperiod started to increase, it is likely that these differences in oestrous behaviour resulted from consumption of Berseem clover isoflavones. The effect of isoflavones depends on the concentration of endogenous E2because isoflavones and E2 are competing for the binding sites on oestrogen receptors (ERs). When there are greater concentrations of endogenous E2 (i.e., follicular phase), isoflavones may function as oestrogenic antagonists inhibiting the full oestrogen activity by occupying a part of the ERs (Lephart et al., 2005). When there are lesser 9

concentrations of endogenous oestrogens, the oestrogenic activity of isoflavones may become manifested (Lephart et al., 2005; Shanle and Xu, 2011). In addition, several phytoestrogens including genistein, daidzein and biochanin A are selective ER modulators that have greater affinity for ERβ than ERα (Lorand et al., 2010).Long-term oestrogen treatment down-regulates the relative abundance of ERα mRNA, but up-regulates the relative abundance of ERβ mRNA of ovariectomized rats (Taleghany et al., 1999). The ERβ receptors are involved in the

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antagonistic action of E2 during seasonal anoestrus in sheep (Romanowicz et al., 2004). When considering these previous findings, the longer time to onset of

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oestrus in Berseem clover-fed ewes may have resulted from the oestrogen

antagonistic actions of the oestrogens in Berseem clover. During the follicular phase, E2concentrations were greatest. With E2 being the dominant steroid, and considering the capacity of isoflavones to up-regulate and bind to theERβ receptor

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functioning as an anti-oestrogen agonist Berseem clover isoflavones might have an

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oestrogenic antagonistic effect that interfered with the endogenous E2 action by

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binding with ERβ or competing with E2 reducing its biological activity. Feeding Berseem clover resulted in a shortened duration of oestrus (Table 4) in

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ewes of the treated group. The duration of oestrous behaviour in sheep depends mostly on the duration of E2 presence rather than on its maximum concentration

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(Fabre-Nys and Gelez, 2007). Following luteolysis, the secretion of E2 increases within 4 to 8 h, after that the secretion rate continues to increase until the time of

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the LH-surge. Within 4 to 8 h of the start of the LH-surge, the secretion of E2 decreases rapidly reaching barely detectable concentratins within 16 h (Campbell et al., 1990). Phytoestrogens can enhance GnRH- induced LH secretion (Arispe et al.,

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2013). Berseem clover isoflavones might, therefore, have advanced the timing of the LH-surge, causing earlier ovulation which might rapidly decrease E2 concentration and thus leading to a shorter period of behavioural oestrus (Hay and

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Moor, 1975).

During the luteal phase, serum P4 concentrations decreased significantly due to Berseem clover feeding (Fig. 3).This finding is consistent with the results obtained by Hashem et al. (2016) where feeding Berseem clover for a long term decreased serum P4 concentration of heifers in early pregnancy (between 7 and 21 days of pregnancy). Also, Nynca et al. (2013) reported that biochanin A inhibited P4 synthesis by granulosa cells of pigs. In addition, Wong and Keung (1999) reported 10

that genistein, daidzein, biochanin A and formononetin inhibited the 3βhydroxysteroid dehydrogenase/isomerase complex (the key enzyme for P4 synthesis from pregnenolone). Generally, isoflavones can alter the concentration and pulse frequency of luteinizing hormone (LH) after ovulation (the luteal phase) that are important for the adequate functionality of corpus luteum (CL) and synthesis of P4. Direct infusion of genistein into the brain of ovariectomised ewes resulted in decreased plasma LH concentrations and LH-pulse frequency (Wojik-

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Gladysz et al., 2005). Also, long term consumption of a soy diet (source of genistein and daidzien isoflavones) inhibits LH-stimulated P4 secretion

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(Wocławek-Potocka et al., 2005).

In the present study, ewes fed Berseem clover or maize silage had comparable conception and lambing rates; however, Berseem clover fed ewes had about 35% less (P = 0.132) fecundity and a 30% smaller (P = 0.085) litter size than maize

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silage fed ewes. Regardless of the statistical insignificance of these varibles, which

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could be due to the small number of ewes used in each experimental group; these

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differences cannot be totally ignored. These results could suggest that feeding Berseem clover might have affected embryonic and/or fetal viability as a result of

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the lesser P4 concentrations that occurred as a result of Berseem feeding in the present study or to the hormonal imbalance during pregnancy. Genistein and

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daidzein stimulated E2 synthesis by human trophoblast (Richter et al., 2009).Furthermore, feeding Berseem clover results in a decrease in serum P4

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concentration and P4/E2 ratio (prevalence of E2) during early pregnancy in heifers (Hashem et al., 2016). The prevalence of E2 during pregnancy has deleterious effects on CL functions and stimulates myometrium contractions, which is

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detrimental to embryo implantation (Jefferson, 2010).

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5. Conclusion

The present study clearly implicated of Berseem clover isoflavones in altering behavioural oestrus of oestrus-induced ewes during the non breeding season by extending the interval to oestrus and shortening oestrous duration. This may be related to the suppressed developmental capacity of small and medium follicles during the follicular phase. Reproductive management throughout the period of 11

feeding Berseem clover, and detection of oestrus has to be more precise so that there will be detection of the largest proportion of females being in oestrus because of the short duration of oestrus in ewes fed Berseem clover. Also, feeding Berseem clover negatively affected luteal progesterone synthesis and this may affect pregnancy outcomes, causing considerable economic loss in sheep industry of Egypt when diets containing this clover are fed.

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Conflict of interest

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None of the authors have any conflict of interest.

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Hashem, N.M., El-Azrak, K.M., Sallam, S.M., 2016.Hormonal concentrations and reproductive performance of Holstein heifers fed Trifolium alexandrinum as a phytoestrogenic roughage. Anim. Reprod. Sci. 170, 121-127.

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Hashem, N.M, El-Azrak, K.M., Nour El-Din, A.N., Taha, T.A., Salem, M.H., 2015. Effect of GnRH treatment on ovarian activity and reproductive performance of low prolific Rahmani ewes.Theriogenology 83, 192-198.

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Hashem, N.M., Sallam, S.M. 2012. Sexual and ovarian activity of crossbred ewes fed different types of roughage during seasonal anoestrus. Small Rumin. Res. 107, 136140.

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Hay, M.F., Moor, R.M., 1975. Functional and structural relationships in the Graafian follicle population of the sheep ovary.J. Reprod. Fertil. 45, 583-593.

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Kolodziejczyk-Czepas, J., Nowak, P., Kowalska, I., Stochmal, A., 2015. Antioxidant action of six Trifolium species in blood platelet experimental system in vitro. Mol. Cell. Biochem. 410,229-237

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Table 1 Chemical composition (g/kg DM), metabolizable energy (ME, MJ/kg DM) and the concentrations of isoflavones (µg/g DM) of the concentrate mixture, Berseem clover and maize silage Concentrate

Chemical composition (g/kg DM) Organic matter Ether extract Crude protein Neutral detergent fibre Acid detergent fibre Acid detergent lignin Cellulose Hemicellulose ME (MJ/kg DM) Isoflavones content1 (µg/g DM) Genistein Daidzein Formononetin Biochanin A Total isoflavones 1 nd= not detected

Experimental diets Berseem clover

A

CC E

PT

ED

M 16

6.60 8.05 2.85 265 282.5

U N

A

0.32 nd nd nd 0.32

849.4 24.7 155.3 497.8 324.7 73.5 251.1 173.1 8.86

SC R

855.9 11.9 126.0 581.8 370.3 127.2 243.1 211.5 6.86

Maize silage 916.3 14.1 76.8 671.7 379.9 54.2 325.7 298.1 7.54

IP T

Parameter

0.20 nd nd nd 0.20

Table 2 Changes in oestrous rate, time to first postpartum ovulation and ovarian activity throughout weeks 2 to 8 postpartum in Rahmani ewes fed Berseem clover (phytoestrogenic roughage) or maize silage (non-phytoestrogenic roughage) during the non-breeding season

1.43 1.17 0.59 0.43 10.07

N A M ED PT CC E A

17

SEM 1.17 0.21 0.18 0.14 0.01 0.06 0.32

Trt

Time (T)

Trt×T

0.952 0.627 0.292

<0.001

0.490

0.851 0.090 0.476 0.407 0.581

0.080 <0.001 0.015 0.031 0.695

0.069 0.729 0.408 0.351 0.346

IP T

1.44 0.87 0.64 0.50 10.17

U

Oestrous rate, % Time to first postpartum ovulation, Days Total number of follicles Follicle distribution Small, ≥2–3 mm Medium, >3–<5 mm Large, ≥5 mm No. of corpora lutea Diameter of corpora lutea, mm

P-value

SC R

Variable

Treatment (Trt) Berseem Maize clover silage 0 0 38.82 34.30 2.96 3.19

Table 3 Changes in ovarian activity during the follicular and luteal phases of CIDR-induced oestrus in Rahmani ewes fed Berseem clover (phytoestrogenic roughage) or maize silage (nonphytoestrogenic roughage) during the non-breeding season

SEM

Trt

4.23 0.87 2.07 1.30

4.37 1.40 2.03 0.93

0.31 0.24 0.24 0.18

0.966 0.156 0.945 0.137

0.044 0.046 0.015 0.001

0.019 0.016 <0.00 1 0.221

1.11 11.24 4.41b

1.13 10.45 6.41a

0.12 2.23 0.62

0.935 0.291 0.023

<0.001

0.003

U

a,b

Within variable means with different superscripts differ (P < 0.05) Follicular phase = day of oestrus and the two preceding days 2 Luteal phase = Days 8, 11 and 18 post-mating

A

CC E

PT

ED

M

A

N

1

18

Time (T)

IP T

Follicular phase1 Total number of follicles Small, ≥2–3 mm Medium, >3–<5 mm Large, ≥5mm Luteal phase2 No. of corpora lutea Diameter of corpora lutea, mm Progesterone, ng/ml

P-value

Treatment (Trt) Berseem Maize clover silage

SC R

Variable1

Trt×T

Table 4 Oestrous characteristics and reproductive performance following CIDR-induced oestrus of Rahmani ewes fed Berseem clover (phytoestrogenic roughage) or maize silage (nonphytoestrogenic roughage) during the non-breeding season SEM

P-value

2.82 2.30 0.28 0.27

0.964 0.004 <0.001 0.488 0.827 0.132 0.085

IP T

Oestrous rate1,% Interval to oestrus2, h Duration of oestrus3, h Conception rate4 at Day 32, % Lambing rate5, % Fecundity6, % Litter size7

Treatment Berseem Maize silage clover 100.00 (12/12) 91.60 (11/12) 57.00a 42.54b 20.00b 34.90a 83.33 (10/12) 90.91 (10/11) 41.67 (5/12) 45.45 (5/11) 58.33 (7/12) 90.91 (10/11) 1.40 (7/5) 2.00 (10/5)

SC R

Variable1

a,b

Within variable means with different superscripts differ (P < 0.05) Oestrous rate = (no. of ewes showing oestrus/total no. of synchronized ewes in each group) ×100 2 Interval to oestrus= the halfway between the time of CIDR removal and the first positive overt signs of oestrus 3 Duration of oestrus = the halfway between the first acceptance of female to be mounted by teaser ram and the last refusal 4 Conception rate = (no. of pregnant ewes at day 32 / no. of inseminated ewes in each group) ×100 5 Lambing rate = (no. of ewes lambing / no. of exposed ewes) × 100.6Fecundity = (no. of born lambs / no. of exposed ewes) × 100 7 Litter size = (no. of born lambs / no. of ewes lambed) × 100

A

CC E

PT

ED

M

A

N

U

1

19

CIDR -out+ eCG Prepartum

Postpartum

Mating

Berseem clover

2 months

8 weeks

8 Days

48 h

32 Days

n=12

Maize silage

2 months

8 weeks

8 Days

48 h

32 Days

n=12

IP T

CIDR-in

Pregnancy diagnosis

A

CC E

PT

ED

M

A

N

U

SC R

Fig.1. Schematic framework to evaluate the effect of feeding Berseem clover (phytoestrogenic roughage) or maize silage (non-phytoestrogenic roughage) on reproductive performance of Rahmani ewes during the non-breeding season (CIDR=controlled internal drug release device, 0.3 g progesterone and eCG= equine chorionic gonadotropin, 500 IU)

20

Berseem clover

Ma ize sila ge

a a

b

IP T

Total number of follicles

6 5.5 5 4.5 4 3.5 3 2.5 2

ab

b

a

3 ab

2 1.5

b

b

A

1 0.5 0

ED

2.5 2

1.5

1

PT

No.of large follicles

a

N

2.5

a

M

No. of medium follicles

3.5

U

No. of small follicles

SC R

a

2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3

0.5

A

CC E

0

day -2

day -1

day 0

Days relativt to oestrus

Fig. 2. Changes in ovarian follicular dynamics during the follicular phase (Day 0 = day of oestrus) of CIDR-induced oestrus in Rahmani ewes fed Berseem clover (phytoestrogenic roughage) or maize silage (non-phytoestrogenic roughage) during the non-breeding season. a,bMeans within variable within treatment with different superscript letters differ (P < 0.05)

21

Maize silage a

14.1 a

12.1

b

a

10.1

b

8.1 6.1

b

4.1 0.1 CIDR removal

24 h

48 h

Day 8

Day 11

Day 18

IP T

2.1

SC R

Progesterone , ng/ml

Berseem clover

Follicular phase

Luteal phase

A

CC E

PT

ED

M

A

N

U

Fig.3. Changes in progesterone concentration (ng/ml) during the follicular phase (day of CIDR removal, 24 and 48h following CIDR removal) and luteal phase (Days 8, 11 and 18 post-mating) of CIDR-induced oestrus in Rahmani ewes fed Berseem clover (phytoestrogenic roughage) or maize silage (non-phytoestrogenic roughage) during the non-breeding season. a,bMeans within the same time point with different superscript letters differ (P < 0.05)

22