Journal Pre-proof

Treatment with eCG and hCG to induce onset of estrous cycles in ewes
during the non-breeding season: effects on follicular development and
fertility

J.H. Dias, V.O. Miranda, F.C. Oliveira, S.F. Vargas Junior, C.S. Haas,
V.G.G. Costa, T. Lucia Jr., A.D. Vieira, C.D. Corcini, B.G. Gasperin

PII: S0378-4320(19)30412-9
DOI: https://doi.org/10.1016/j.anireprosci.2019.106232
Reference: ANIREP 106232

To appear in: Animal Reproduction Science

Received Date: 7 May 2019

Revised Date: 21 September 2019
Accepted Date: 8 November 2019

Please cite this article as: Dias JH, Miranda VO, Oliveira FC, Vargas SF, Haas CS, Costa
VGG, Lucia T, Vieira AD, Corcini CD, Gasperin BG, Treatment with eCG and hCG to induce
onset of estrous cycles in ewes during the non-breeding season: effects on follicular
development and fertility, Animal Reproduction Science (2019),
doi: https://doi.org/10.1016/j.anireprosci.2019.106232

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Treatment with eCG and hCG to induce onset of estrous cycles in ewes during the

non-breeding season: effects on follicular development and fertility

J.H. Dias¹, V.O. Miranda†¹, F.C. Oliveira¹, S.F. Vargas Junior¹, C.S. Haas¹, V.G.G.

Costa¹, T. Lucia Jr.¹, A.D. Vieira¹, C.D. Corcini¹, B.G. Gasperin¹*

¹ReproPEL, Departamento de Patologia Animal, Faculdade de Veterinária,

Universidade Federal de Pelotas, Campus Universitário s/n, CEP: 96010-900, Capão

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do Leão, Rio Grande do Sul, Brasil

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†In memoriam

*
Corresponding Author: bggasperin@gmail.com
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ABSTRACT
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Although combining of eCG and hCG administrations is known to enhance LH-like

actions, there have been few studies where there was comparison of the effects of
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treatment of anestrous ewes with eCG and hCG and eCG alone. In Experiment 1, 18

ewes in seasonal anestrus were administered an intravaginal device (IVD) containing

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medroxyprogesterone acetate for 12 days, and at the time of IVD removal (D0), were
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allocated into the following groups (n = 6/group): no further treatment (control); 400 IU

eCG (eCG); or 400 IU eCG and 200 IU hCG (eCG+hCG). There was greater ovarian

follicular growth in the groups treated with gonadotropins, compared to the control, and

there were greater progesterone concentrations in the eCG+hCG group on D9 (P<0.05).

In Experiment 2, 66 ewe lambs were assigned to the same treatment groups described

for Experiment 1, and subsequently there was natural mating with rams. There was a

1
greater rate of behavioral estrous manifestation in the eCG (88.5%; 23/26) and

eCG+hCG (85.2%; 23/27), than control (30.8%; 4/13; P<0.05) group. Pregnancy rate

was also greater in the eCG (34.6%; 9/26) and eCG+hCG (18.6%; 5/27) than control

(0%; 0/13; P<0.05) group, whereas conception rate, considering only ewe lambs that

were mated, was only greater in the eCG group. Although there were greater

progesterone concentrations 9 days after treatment in the eCG+hCG group, there was no

difference in follicular growth in anestrous ewes, nor was there an effect on estrous

behavior manifestation and pregnancy rates in ewe lambs, compared to treatment with

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only eCG.

Keywords: Ewe lambs; Follicular growth; Hormonal protocol.

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1. Introduction
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Due to the increased demand for sheep products such as wool, meat and milk,
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farmers are interested in new technologies to address supply challenges and that allow

for greater production of these products. Breeding seasonality in small ruminants is a

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limiting factor to production expansion (Fatet et al., 2011). The use of hormonal

treatments, therefore, is a viable alternative to induce onset of estrous cycles, reducing

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the lambing interval and subsequently increasing production efficiency of sheep meat,

wool, and milk. During anestrus, treatment regimens based on progestins combined
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with gonadotropins are recommended for use because progesterone (P4) controls LH

secretion, whereas gonadotropins directly stimulate follicular growth, estrous behavior

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manifestation and ovulations of eggs with the capacity for fertilization in anestrous

females (Abecia et al., 2012).

In ruminants, eCG binds mainly to the FSH receptor, but also to the LH receptor

(LHCGR), stimulating follicular growth (Murphy, 2012). Use of eCG during the non-

2
breeding season improves the rate and extent of behavioral estrous expression in ewes

and, therefore, there is a greater ovulation response (Kridli and Al-Khetib, 2006).

Human chorionic gonadotropin (hCG) has been used to induce ovulation in

several species because it binds to the LHCGR receptor and has a marked luteotropic

activity, thus, when there is treatment with hCG there is a greater P4 secretion. In cows

(De Rensis et al., 2008), and ewes (Gómez-Brunet et al., 2007), the administrations of

hCG treatments has led to increased pregnancy rates in females previously having a

greater-than-expected incidence of embryonic losses or compromised fertility. There is

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frequently administration of eCG combined with hCG in gilts and sows because there is

a combined product available is some countries (Breen et al., 2006) and this is an

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alternative in those countries where there is not an eCG alone that is commercially
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available. Even though hCG administration potentially results in greater LHCGR

activation and, consequently, a likely greater synchrony in time when ovulations occur
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and greater luteal function, it is not completely understood if treatment with hCG results
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in an improved response to eCG in ewes, especially during seasonal anestrus. In

previous studies with ewes, there was an evaluation of the behavioral estrous response

and time of ovulation relative to time of progestin treatment cessation (Quirke et al.,
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1979; Cline et al., 2001).

Based on the results from previous studies, it was hypothesized that

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administering eCG+hCG at the end of a progestin treatment period will improve rate of
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follicular growth, synchrony in time ovulation occurs among ewes and luteal function,

compared with the treatment with eCG alone. The objective of the present study,

therefore, was to assess the reproductive outcome when there was treatment with 400 IU

eCG, without administration of hCG or by combining this eCG treatment with

administration of 200 IU hCG, to induce estrus and ovulation in anestrous ewes.

3
2. Materials and methods

All procedures were approved by the Ethics Committee in Animal

Experimentation of Universidade Federal de Pelotas (7340-2016).

2.1. Experiment 1: Effect of eCG+hCG treatment on follicular dynamics and luteal

function in anestrous ewes

2.1.1. Animals and estrous synchronization protocol

The experiment was conducted at a commercial farm (28°S 55°W), in November

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during the non-breeding season in the southern hemisphere. Polwarth ewes (n = 18),

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with a maximum age of 24 months and an average body condition score of 3 (on a 1 to

5 scale), evaluated as described in Kenyon et al. (2014), were assigned to groups with
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there being ovulation induction and ovarian follicular dynamics assessments. Ewes were

maintained in pastures containing native forage and had free access to water. All ewes
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were administered an intravaginal device (IVD; D-12) containing 60 mg
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medroxyprogesterone acetate (MPA), which remained in place for 12 days. At the time

of IVD removal from the vagina (D0), ewes were assigned to one of three groups (n =
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6/group): control, which received no further treatment after the removal of the IVD;

eCG, which were administered 400 IU eCG IM (Novormon®, Zoetis, Campinas, SP,
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Brasil) at the time of IVD removal; and eCG+hCG, which were administered 400 IU

eCG and 200 IU hCG IM (Chorulon®, MSD Saúde Animal, São Paulo, SP, Brazil) at
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the time of IVD removal.

2.1.2. Ultrasonic assessments

Follicular growth evaluations were performed in all females using a B-mode

ultrasonography instrument (Mindray DP-2200 VET®, Shenzhen, China) equipped with

4
a 7.5-MHz transrectal linear transducer stiffened with a hollow aluminum rod.

Evaluations began 24 h after IVD removal (in the control group) and after IVD removal

and gonadotropin administrations in the two groups in which these treatments were

imposed and were performed every 12 h for 4 days. Follicle diameter, position of

follicles in the ovary, assessment of follicular growth rate and regression patterns

(mm/day) were determined and values for these variables were recorded. Ovulation was

defined as to have occurred when there was disappearance, based on ultrasonic

assessments, of a previously identified pre-ovulatory follicle.

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2.1.3. Blood sampling

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Blood samples were collected, using venipuncture of the jugular vein, into
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vacuum tubes (BD Vacutainer®, São Paulo, SP, Brazil) on the day of IVD insertion (D-

12), and 9 days after IVD removal (D9). Samples were centrifuged at 1,500 x g for 10
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minutes, serum was decanted and stored at -20 ºC, and the serum P4 concentrations
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were assessed using chemiluminescence procedures (ADVIA Centaur; Siemens; Ref.

01586287; sensitivity of 0.21 µg/L; analytical measuring range: 0.21 µg/L to 60.00

µg/L), with intra- and inter-assay values of less than 10%. The assay was previously
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validated and used for quantifying concentrations of progesterone in sheep (Miranda et

al., 2018).
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2.2. Experiment 2: Effect of eCG+hCG treatment on estrous manifestation, luteal

function and percentage pregnancy rates

2.2.1. Animals and estrous synchronization treatment regimens

The experiment was conducted at a commercial farm (31°S 52°W) from

September to October, during the non-breeding season for sheep in the southern

5
hemisphere. In Polwarth ewe lambs (n = 66), aged 12-months, there was imposing of an

ovulation induction treatment regimen. All animals were placed in a pasture containing

native forages and had free access to water. The treatment regimen for estrous

synchronization was that which was previously described in this manuscript for

Experiment 1. At the time of IVD removal (Hour 0), ewes were assigned to one of three

groups: control (n = 13); treatment with eCG (n = 26); and treatment with eCG+hCG (n

= 27). Immediately after the gonadotropin treatments were administered to ewes of the

two groups or in the control group there was removal of the IVD, the ewe lambs were

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placed with rams for the purpose of natural mating. There was a breeding soundness

evaluation of rams before they were used for breeding and there was a 1:15 ram:ewe

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ratio utilized for breeding. Pregnancy diagnosis was performed approximately 25 days
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after the expected time of ovulation (D28) by performing a transrectal ultrasonic

examination (Sonoscape A5 Vet) utilizing a 7.5-MHz transrectal linear transducer

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stiffened with a hollow aluminum rod. Conception rate was defined as the proportion of
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ewe lambs pregnant out of those that were marked by the rams and mated. Pregnancy

rate was defined as the proportion of ewe lambs pregnant out of those assigned to each

group.
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2.2.2. Monitoring of estrous behavior manifestation

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To confirm whether mating occurred, rams were equipped with a marking

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harness. Estrous behavior manifestation was monitored every 4 h, for 120 h from the

time when rams were placed with the ewe lambs (Hour 0). Ewe lambs marked with

paint were considered to be or to have been in estrus, and females with markings

indicating mating had occurred were separated from the flock every 8 h.

6
2.2.3. Blood sampling

Blood samples were collected on the day of IVD insertion (D-12), to ascertain

whether ewes were seasonally anestrus at the beginning of the study and again 9 (D9)

and 14 (D14) days after IVD removal. The blood collection procedure and P4 assay

were conducted using procedures described for Experiment 1.

2.3. Statistical analysis

In Experiment 1, data for P4 concentrations on D9 were compared among

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treatment groups using an analysis of variance (ANOVA) utilizing JMP software. Data

for follicular dynamics were analyzed using the MIXED procedure of the SAS®

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statistical software utilizing a repeated measure statement. The main effects of treatment
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group, day and the interaction were included in the statistical model. Differences

between follicular sizes at specific time points were compared among groups using a
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Student’s t-test utilizing least squares means. Although different covariance structures
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were evaluated, the autoregressive option was selected because with it there was the

least Akaike Information Criteria (AIC). In Experiment 2, the effect of treatment on

estrous behavior expression, pregnancy percentage and conception percentage rates was
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evaluated using the chi-square test, by means of a generalized linear model fit for a

binomial distribution and contrast as a post-hoc test, using JMP® software. Due to lack
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of normality (as indicated by the Shapiro-Wilk test), progesterone concentrations on D9

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and D14 were log transformed and then compared among treatment groups and periods

using an ANOVA with repeated measures using Statistix® software, with the individual

effect nested within treatments.

3. Results

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3.1. Experiment 1

All of the ewes were seasonally anestrus at the time of initiation of the study as

based on the finding that P4 concentrations were less than 1 ng/mL in all ewes included

in the present study. There was greater follicular development at 36 and 48 h after IVD

removal from the vagina of ewes of the eCG and eCG+hCG groups, with these ewes

having a larger follicular diameter (P<0.05) than the ewes of the control group (Fig. 1).

With both gonadotropin treatments (eCG and eCG+hCG groups), ewes had more

follicles with a mean diameter of greater than 6 mm at 48 h after IVD removal, whereas

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the control group had an average follicle diameter of 4.8 mm at this time point. There

was an effect of time (P<0.01) on follicular growth, indicating there was a different

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pattern of follicular growth among ewes in the different treatment groups. The treatment

by period interaction was not significant (P = 0.5).

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Data for ovulation rates are shown in Table 1. In the control group, only two
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ewes had ovulations (2/6), and ovulations occurred after 84 h from the time of IVD
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removal from the vagina. In the eCG treatment group, all six ewes had ovulations after

removal of the IVD and eCG treatment, with ovulations occurring at about 60 h after

removal of the IVD and eCG treatment, with two exceptions where there were
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ovulations detected to have occurred at the 72 h assessment period. There were two

ovulations occurring in each of three animals of the eCG treatment group, therefore,
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there were a total of nine follicles from which ovulations occurred in this group. In the
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eCG+hCG treatment group, there were ovulations in five ewes (5/6), and the time of

ovulation was variable, with there being two ovulations detected at 60, four at 72, and

two at 84 h after removal of the IVD from the vagina and treatment with eCG and hCG.

There were two or three ovulations detected in two ewes of the eCG+hCG group, thus,

there was a total of eight follicles from which ovulation occurred in this group. There

8
were differences among groups in serum P4 concentrations on D9 (P<0.05), with

concentrations in the ewes of the eCG+hCG treatment group being greater than in the

other two groups (Fig. 2).

3.2. Experiment 2

All of the ewe lambs were in seasonal anestrus at the start of the treatment

period, based on there being P4 concentrations of less than 1 ng/mL in all ewe lambs. A

total of 50 animals (75.6%) expressed symptoms of estrous behavior as a result of the

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treatments that were imposed, of which 30.8% (4/13), 88.5% (23/26) and 85.2% (23/27)

were from the control, eGC and eCG+hCG groups, respectively. There were more ewes

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in the two gonadotropin treatment groups (eGC and eCG+hCG groups) expressing
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symptoms of behavioral estrus than in the control group (P<0.05).

The period during which there was expressions of estrous behavior in the
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eCG+hCG treatment group ranged from 31 and 71 h (Fig. 3). In the eCG group, all
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ewes expressed symptoms of estrous behavior before 51 h from the time of IVD

removal and administration of the eCG treatment, whereas behavioral estrous symptoms

in ewes of the control group only occurred after 51 h from the time of IVD removal.
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The eCG treatment group had the greatest cumulative frequency of ewes in estrus at 51

h (88.5%) after removal of the IVD, compared with ewes of both the eCG+hCG
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(55.5%) and control (16.6%) group (P<0.05).

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Pregnancy percentage rate (Fig. 4) was greater in the ewe lambs of the eCG

(34.6%, 9/26) and eCG+hCG (18.6%, 5/27) groups, than the control (0%; 0/13) group

(P<0.05). Conception rate was greater in the ewe lambs of the eCG (39.1%, 9/23) group

in comparison to the control (0%; P<0.05), whereas the conception rate in ewes of the

eCG+hCG group (21.7%, 5/23) did not differ from the other groups (P>0.05). There

9
was an increase in serum P4 concentrations between D9 and D14 after IVD removal,

however, there was no effect of treatments or of the treatment by period interaction on

concentration of progesterone during the luteal phase after IVD removal from the

vagina (Fig. 5).

4. Discussion

Gonadotropin supplementation is effective for promoting follicular growth and

stimulating onset of estrous cycles in ewe lambs or ewes during the non-breeding

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season. Supplementation commonly occurs with the administration of eCG, a molecule

with a binding affinity for FSH and LH receptors (Murphy, 2012). In some countries

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where eCG alone is not commercially available, the only alternative for administration
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of eCG is in a combination dose of eCG and hCG because in these countries there is a

combined eCG and hCG product that is commercially available for use in swine.
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Although both approaches are considered to be effective, information about which
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treatment provides the most desirable response in anestrous ewes is minimal (Quirke et

al., 1979; Cline et al., 2001). In the present study, the imposing of the eCG+hCG

combined treatment resulted in follicular growth patterns similar to that when there was
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imposing of the eCG treatment alone. Even though there was a lesser synchrony in the

time of ovulation with use of the eCG+hCG treatment, there were greater serum P4
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concentrations in ewes from the eCG+hCG group 9 days after the removal of the IVD
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from the vagina and administration of the eCG and hCG treatments. There was an

induced estrus and ovulation in ewe lambs in both the eCG and eCG+hCG groups,

whereas there were greater pregnancy and conception rates in the ewes of the eCG, but

not those of the eCG+hCG group, compared to non-treated control animals.

10
 In Experiment 1, the use of eCG+hCG resulted in follicular growth promotion in

anestrous ewes, as expected. Post treatment with eCG and hCG, however, the time of

ovulation was quite variable, occurring after the majority of ewes from the eCG group

had already had ovulations. To the best of our knowledge, there are only two studies

comparing the eCG with eCG+hCG treatment in anestrous ewes (Quirke et al., 1979;

Cline et al., 2001) and in these two studies there was only evaluation of the estrous

response and time of ovulation subsequent to treatments being imposed. Quirke et al.

(1979) reported that there was a greater proportion of ewes in estrus but a lesser

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ovulation rate after eCG treatment compared to treatment with eCG+hCG. In the study

of Cline et al. (2001), treatment of ewes with eCG and hCG resulted in a delayed

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ovulation and decreased synchrony in timing of ovulation compared to what occurred in
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ewes treated with eCG alone. Using a progestagen plus eCG protocol, Barret et al

(2004) reported that there was no difference in the frequency of ovulations occurring
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during either estrous or anestrous periods, with a maximum diameter of pre-ovulatory
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follicles of 6.1 mm, similar to the results in the current study when there was use of the

same treatment.

Interestingly, in Experiment 1, although there were the least, as compared with

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the other two groups, P4 concentrations in the ewes of the control group, as expected,

in the ewes of the eCG+hCG group there were greater P4 concentrations 9 days (D9)
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subsequent to IVD removal. Administration of both eCG and hCG can result in greater
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P4 concentrations due to the luteotropic effects of these two gonadotropins (Gómez-

Brunet et al., 2007; Souza et al., 2009). It has also been reported that hCG could

promote increased blood circulation in the corpus luteum, which may be related to the

increase in circulating P4 concentration (Zygmunt et al., 2002). The relatively greater

P4 concentrations in the ewes treated with eCG and hCG may positively affect

11
pregnancy maintenance in embryo recipients, although this hypothesis still needs to be

tested.

In Experiment 2, greater than 80% of the ewe lambs from both groups treated

with gonadotropins expressed symptoms of estrous behavior and there was no

difference between the two groups in this regard. Ewe lambs of the control group

treated with MPA alone had very little manifestation of behavioral estrous symptoms.

These results corroborate those of D'Souza et al. (2014) in ewes, and Knight et al.

(2015), in ewe lambs, where there was lesser estrous behavior expression in ewes

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treated with P4 alone (79.2% and 52.6%, respectively) compared with ewes treated with

P4, eCG, and hCG (94.2% and 77.9%, respectively). Knight et al. (2015), however,

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reported that treatment with P4 alone resulted in induction of onset of estrous cycle in
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lambs, which is inconsistent to what was observed in the present study. Because both

such studies were conducted in the USA, treatment with eCG alone was not evaluated.
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Although the estrous behavior manifestation rates did not differ between the
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gonadotropin-treated groups in the present study, there was more variation in the time

of estrous behavior expression subsequent to administration of the eCG combined with

hCG treatment as compared to that of ewe treated with eCG alone. This finding is
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consistent with those reported in a previous study of Cline et al. (2001) where there was

use of the same treatment regimens in ewes. Nevertheless, in the previous study, the
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IVD that was used contained norgestomet rather than MPA or P4, and even though the
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behavioral estrous rates and synchrony in time of expression of estrous behavior were

assessed, the fertility of the ewes was not evaluated. Santos et al. (2010) also reported

that there was a later onset of estrus after treatment with an eCG and hCG treatment

regimen compared to what occurred in ewes treated with only MPA or MPA combined

with eCG. In this previous study, there was a behavioral estrous rate of 73.3% in ewes

12
treated only with MPA IVDs, thus, these results are inconsistent with those in the

present study. Because there were no P4 quantification assessments in this previous

study, it is possible that some ewes had already initiated onset of estrous cycling prior to

the treatment period. Inconsistent with the data in ewes, imposing a combined eCG and

hCG treatment regimen in goat does resulted in a lesser period from the time treatments

were administered to estrous onset and a greater pregnancy rate compared with

treatment with eCG alone (Rowe and East, 1996). In results from this previous study in

which the eCG+hCG treatment in goats was also evaluated during the seasonal

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transitional period from anestrus to onset of estrous cycles, there was a synergistic effect

of these two gonadotropins in reducing the interval from imposing the treatment to the

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time when estrus occurred and there was a positive effect on percentage fertility rates.
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Both eCG and hCG are protein hormones with α and β subunits, large molecular

weights, and a large amount of glycosylation, that when administered have a long half-
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life in blood plasma: 21 h for eCG in ewes; 39.4 h for hCG in goat does (Murphy, 2012;
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Saleh et al., 2012). While eCG functions to promote follicular growth, hCG functions to

induce luteinization of follicular cells of any size that have LHCGR, and possibly has

actions that result in ovulations from immature follicles (Bartlett et al., 2009). Based on
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the results from the current and previous studies, it can be hypothesized that as a result

of the long half-life of hCG there is a longer period of hCG-induced follicle growth
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preceding the time of ovulation, including in follicles that are not responsive at the time
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when eCG is administered alone without inclusion of hCG as part of the treatment

regimen. This hypothesis, however, still needs to be investigated.

Administration of ovulation inducers usually occurs at the time of withdrawal of

the exogenous progestagen source, a few hours before artificial insemination (AI) or

even at the time of AI. Gomez-Brunet et al. (2007) administered 500 IU hCG at the time

13
of artificial insemination in a P4/eCG treatment regimen, and there were improved

reproductive responses only at those farms where reproductive rates were sub-optimal

based on farm records that were accrued before the study was conducted.

Administration of hCG 36 h after removal of the IVD combined with eCG

administration did not have an effect on ovulation and pregnancy rates (Khan et al.,

2003) and on P4 concentrations when there were comparisons between the treated and

control groups of ewe lambs (Catalano et al., 2012).

In the present study, pregnancy rates in ewe lambs were greater in groups where

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there was treatment with eCG or eCG combined with hCG compared to the control

group, although there was a greater conception rate only after treatment with eCG alone.

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Similarly, Santos et al. (2010) reported that pregnancy rates in artificially inseminated
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ewes after eCG treatment were greater than when there was treatment with eCG and

hCG. It is possible that a lack of precision in determining the time of ovulation or

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ovulatory failures in the eCG+hCG group could be involved in this lesser fertility,
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because in the present study there was a large difference between the percentage of

ewes expressing behavioral estrus (85.2%) and pregnancy rate (18.6%). Another

possibility would be the occurrence of an early regression of the CL, a phenomenon that
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occurs after treatment with eCG, hCG and GnRH, which is associated with a less-than-

optimal follicular and luteal phase concentration of estradiol and P4, respectively
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(Rubianes et al., 1996), resulting in failure of ovulations from dominant follicles. In the
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present study, P4 concentrations remained at typical luteal phase concentrations on D14,

indicating there was no premature luteal regression in ewes expressing behavioral

estrus. It is noteworthy that the peak of pre-ovulatory LH surge release that induces

ovulation at estrous onset in ewe lambs is less than in mature ewes (Davies and Beck,

14
1993). Furthermore, it is suggested that ovaries of ewe lambs are less responsive to hCG

compared with the ovaries of multiparous ewes (Khan et al., 1998).

5. Conclusion

Although the eCG+hCG treatment in the present study induced follicular growth

and estrus in ewes, the lesser synchrony in timing when ovulations occurred may limit

its use when there is imposing of fixed-time artificial insemination treatment regimens.

The treatment with eCG and hCG in combination resulted in greater P4 concentrations 9

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days post-treatment and this may be beneficial for pregnancy maintenance. In ewe

lambs, although with the imposing of the eCG+hCG treatment regimen there was an

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adequate estrous response, there was a greater conception rate when there was use of

eCG alone.
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Acknowledgments

The authors are thankful to the Conselho Nacional de Desenvolvimento

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Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio

Grande do Sul (FAPERGS – Edital PRONEX 12/2014 -FAPERGS/CNPq, 16/2551-

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0000494-3), and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES; Financial code 001), for the financial support given to this study. C.D. Corcini
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(Process no. 310203/2018-0), T. Lucia Jr. (Process no. 303559/2015-2) and B.G.

Gasperin (Process no. 309138/2017-5) are research fellows from CNPq.

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15
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in small ruminants. Anim. Reprod. Sci. 130, 173-179.

Barrett, D., Bartlewski, P., Batista-Arteaga, M., Symington, A., Rawlings, N., 2004.

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Figure legends

Fig. 1. Follicular growth in anestrous ewes treated with three different ovulation

induction regimens; Anestrous ewes were assigned to the following groups (n =

6/group): MPA-intravaginal device (IVD) for 12 days with no further treatment

(control); 400 IU eCG (eCG); or 400 IU eCG+200 IU hCG (eCG+hCG) at IVD removal

from the vagina (0 h); Asterisks indicate differences among groups (control compared

with eCG and eCG+hCG groups; P<0.05)

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Fig. 2. Serum progesterone concentration (ng/mL) on D9 after MPA- intravaginal

device (IVD) removal from the vagina (D0); Anestrous ewes were assigned to the
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following groups (n = 6/group): IVD for 12 days with no further treatment (control);
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400 IU eCG (eCG); or 400 IU eCG+200 IU hCG (eCG+hCG) at IVD removal (0 h);
a,b
Different letters indicate difference among groups (P<0.05)

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 of
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Fig. 3. Cumulative behavioral estrous detections after ram placement with ewes; Ewe

lambs were assigned to the following groups: MPA- intravaginal device (IVD) for 12
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days with no further treatment (control; n = 13); 400 IU eCG (eCG; n = 26); or 400 IU

eCG+200 IU hCG (eCG+hCG; n = 27) at the time of IVD removal from the vagina (0
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h)
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Fig. 4. Percentage pregnancy rate of ewe lambs treated with three different ovulation

induction treatment regimens; Ewe lambs were assigned to the following groups: MPA-

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intravaginal device (IVD) for 12 days with no further treatment (control; n = 13); 400

IU eCG (eCG; n = 26); or 400 IU eCG+200 IU hCG (eCG+hCG; n=27) at time of IVD

removal from the vagina (D0); a,bDifferent letters indicate difference among groups

(P<0.05)

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Fig. 5. Serum progesterone concentration (ng/mL) on days D9 and D14 after MPA-

intravaginal device (IVD) removal from vagina (D0) of ewe lambs treated with three
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different ovulation induction treatment regimens; Ewe lambs were assigned to the

following groups: IVD for 12 days with no further treatment (control; n = 13); 400 IU
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eCG (eCG; n = 26); or 400 IU eCG+200 IU hCG (eCG+hCG; n = 27) at time of IVD

removal from the vagina (D0).

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Table 1
Ovulation rate of anestrous ewes treated with three different hormonal regimens

Treatment Follicles from which there were ovulations Ewes Ovulating1

at each period (n)*
60h 72h 84h 96h
Control 1 1 2/6 (33.3%)
eCG 7 2 6/6 (100%)
eCG+hCG 2 4 2 5/6 (83.3%)
Anestrous ewes were assigned to the following groups (n = 6/group): MPA-

impregnated intravaginal device (IVD) for 12 days with no further treatment (control);

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400 IU eCG (eCG); or 400 IU eCG+200 IU hCG (eCG+hCG) at time of IVD removal

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(0 h). *Ewes having two ovulations were observed in three animals from eCG group,

whereas there were two or three ovulations detected in two ewes of the eCG+hCG
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group; 1Number of ewes that had ovulations during the observation period.
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