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Animal Reproduction Science 144 (2014) 90– 94

Contents lists available at ScienceDirect

Animal Reproduction Science

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

etection of estrous behavior in buffalo heifers byadiotelemetry following PGF2� administration during thearly or late luteal phase

.M. Porto-Filhoa, L.U. Gimenesb, B.M. Monteiroa, N.A.T. Carvalhoc,.P.S. Ghumand, E.H. Madureiraa, P.S. Baruselli a,∗

Departamento de Reproduc ão Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Sãoaulo, BrazilDepartamento de Medicina Veterinária Preventiva e Reproduc ão Animal, Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal,

ão Paulo, BrazilUnidade de Pesquisa e Desenvolvimento de Registro-Pólo Regional do D.S.A. do Vale do Ribeira/APTA, Registro, São Paulo, BrazilDepartment of Veterinary Gynaecology and Obstetrics, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciencesniversity, Ludhiana, Punjab, India

a r t i c l e i n f o

rticle history:eceived 16 October 2013eceived in revised form3 December 2013ccepted 18 December 2013vailable online 31 December 2013

eywords:adiotelemetryuffalo heifersrostaglandinynchronizationstrus

a b s t r a c t

This study examined the usefulness of radiotelemetry for estrous detection in buffalo heifersand the impact of prostaglandin F2� (PGF2�) administration during the early or late lutealphase on estrous behavior and ovulatory follicle variables. Induction of estrus with PGF2�

at a random stage of the estrous cycle was followed by the arbitrary division of heifersinto groups receiving a second dose of PGF2� during either the early (n = 33) or late (n = 17)luteal phase (6–9 or 11–14 days after estrus, respectively) for the induction of synchronizedestrus. The electronic detection of synchronized estrus by radiotelemetry was confirmedusing ultrasonography every 6 h until ovulation. Radiotelemetry was 90% efficient and 100%accurate for estrous detection. Intervals between the PGF2� dose and the beginning of syn-chronized estrus (40.7 ± 10.9 vs. 56.7 ± 12.8 h) or ovulation (70.0 ± 11.3 vs. 85.6 ± 12.5 h)were shorter (P < 0.05) for heifers receiving PGF2� during the early luteal phase. PGF2�

administration during the early or late luteal phase produced similar (P > 0.05) results forthe duration of estrus, the intervals from the beginning or end of estrus to ovulation, thenumber and duration of mounts per estrus, the duration of mounts, the diameter of the ovu-

latory follicle and the luteal profile on day 5 after estrus. In conclusion, radiotelemetry wasa suitable tool for the efficient and accurate detection of estrus in buffalo heifers. Treatmentwith PGF2� during the early luteal phase had a shorter interval to synchronized estrus andovulation; however, estrous behavior, ovulatory follicle dynamics and subsequent luteal

ar follo

activity were simil

∗ Corresponding author at: Av. Prof. Dr. Orlando Marques de Paiva, 87,5508-270, Cidade Universitária, São Paulo, SP, Brazil.el.: +55 11 3091 7674; fax: +55 11 3091 7412.

E-mail address: barusell@usp.br (P.S. Baruselli).

378-4320/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.anireprosci.2013.12.006

wing PGF2� administration during the early or late luteal phase.© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The efficiency of reproductive biotechnologies, such asartificial insemination (AI), for buffalo herds based onlyon visual estrous detection is seriously compromised due

to the discrete estrous behavior of the buffalo species,especially among buffalo heifers (Baruselli et al., 1997a;Campanile et al., 2010; Jacomini, 1989; Singh et al., 1984;Vale and Ribeiro, 2005; Zicarelli et al., 1997). Estrus

eprodu

R.M. Porto-Filho et al. / Animal R

synchronization with prostaglandin F2� (PGF2�) is aneffective and economical tool for improving the estrousdetection efficiency and use of AI in buffaloes (Chauhanet al., 1985; Diaz et al., 1994; Jha, 2011; Mirmahmoudiand Prakash, 2012). Ovulation can occur up to 6 days afterPGF2� administration, depending on the responsivenessof the corpus luteum (CL) and the stage of ovarian fol-licle development at the time of PGF2� administration(reviewed in Bó et al., 2003).

However, knowledge about buffalo estrous behaviorand ovarian activity following PGF2� administration dur-ing the luteal phase of the estrous cycle is unavailableand is essential for the successful application of reproduc-tive technologies in this species. Moreover, studies are notavailable regarding the practicality of radiotelemetry, anelectronic estrous detection system, in buffalo heifers.

Thus, the objective of this study in buffalo heifers wasto determine the usefulness of radiotelemetry for estrousdetection as well as to observe the impact of PGF2� admin-istration during the early or late luteal phase on theinduction of estrous behavior, dominant follicle dynamicsand subsequent luteal activity. We hypothesized that theinterval between induced estrus and ovulation would beshorter in buffalo heifers treated with PGF2� during theearly luteal phase and that PGF2� treatment during theearly or late luteal phase would not interfere with estrouscharacteristics, dominant follicle development and subse-quent CL function.

2. Materials and methods

2.1. Animals

Fifty cycling Mediterranean buffalo heifers between 24and 30 months of age were maintained on a Brachiariadecumbens pasture with mineral supplementation fromApril to July. The experiment was conducted at the Centrode Biotecnologia em Reproduc ão Animal do Departamentode Reproduc ão Animal da Faculdade de Medicina Veter-inária e Zootecnia da Universidade de São Paulo – Campusde Pirassununga (São Paulo, Brazil; latitude 21◦59′ South,longitude 47◦26′ West).

2.2. Estrus synchronization and PGF2˛ administration

To synchronize the estrus of buffalo heifers (n = 50),a luteolytic dose of a PGF2� analogue (15 mg luprostiol;Prosolvin®) was administered intramuscularly at a randomstage of the estrous cycle. The day of estrous expression fol-lowing the first PGF2� dose was recorded and all heifersreceived a second intramuscular dose of PGF2� (15 mgluprostiol) either in the early (6–9 days after estrus; n = 33)or late (11–14 days after estrus; n = 17) luteal phase. Theday of estrous expression by buffalo heifers following the

second PGF2� dose was considered the day of synchronizedestrus (D0) and was used for describing the characteristicsof estrous behavior recorded by radiotelemetry as well asthe related ovarian activity.

ction Science 144 (2014) 90– 94 91

2.3. Estrous detection by a radiotelemetry system

Each heifer had a transmitter (HeatWatch®, DDx, Incor-porated, Boulder, CO, USA) with a 0.4 km range fixed toits rump. The pressure-sensitive sensor was activated bythe weight of a mounting female, which sent a radiotele-metric signal to a microcomputer via a radio antenna fixedadjacent to the pasture. Two androgenized buffalo cowsreceiving an initial dose of 2000 mg of testosterone propio-nate (Durateston®; Intervet, Lane Cove, NSW, Australia),followed by 1000 mg every 15 days until the end of theexperiment, were also used for the detection of estrus.All information regarding heifer identification, date, timeand the duration of mounts were recorded and processedby the transmitter software (HeatWatch TR-1 ThermicroniButton).

The radiotelemetry system was set to identify the begin-ning of synchronized estrus when there were at least twomounts (≥2 s duration) in a one-hour period and the end ofestrus was determined by an interval of ≥4 h between twoconsecutive mounts. The estrous behavior was classifiedas high (≥1.5 mounts/h) or low (<1.5 mounts/h) intensityand as short (<7 h) or long (≥7 h) duration (Dransfield et al.,1998). The beginning of estrus and mounting behavior wereclassified according to the period of the day, defined asmorning (6:01 h to 12:00 h), afternoon (12:01 h to 18:00 h),night (18:01 h to 00:00 h) and dawn (00:01 h to 6:00 h).

2.4. Ultrasonographic examinations

Transrectal ovarian ultrasonographic examina-tions were performed using a linear probe of 5.0 MHz(AlokaSSD500 micrus®; Japan). The first examination ofheifers was performed 12 h after the beginning of synchro-nized estrus, followed by every 6 h until ovulation. Thetime of ovulation was recorded as the mean hour betweenthe examination in which ovulation was detected and theprevious one.

2.5. Blood collection and hormone assays

Jugular blood samples were collected in 10 mL EDTA-treated tubes (Vacutainer, Becton Dickinson and Company,USA) on the day of the second PGF2� dose, on the day ofthe onset of synchronized estrous (D0) and on day 5 afterestrus. The blood samples were promptly centrifuged at1500 × g and the plasma was stored at −20 ◦C. The lutealactivity was determined by assessing the concentrations ofplasma progesterone using a solid-phase radioimmunoas-say (Coat-a-Count®, Diagnostic Products Corporation, LosAngeles, CA, USA).

2.6. Statistical analysis

The efficiency and accuracy of the radiotelemetry sys-tem for the detection of estrus was confirmed by theultrasonographic detection of ovulation and by the plasma

progesterone concentrations. The efficiency was definedas the percentage of estruses identified by radiotelemetryout of the total number of estruses detected (a measure ofthe false negatives) and the accuracy was defined as the

92 R.M. Porto-Filho et al. / Animal Reproduction Science 144 (2014) 90– 94

Table 1Mean values by treatment group (early or late luteal phase) of estrous behavior, ovulatory follicle dynamics and luteal activity in buffalo heifers.

Variables ELPa LLPb P

Number of buffalo heifers 33 17 –Interval PGF2� to beginning of estrus (h) 40.7 ± 10.9 56.7 ± 12.8 <0.05Interval PGF2� to ovulation (h) 70.0 ± 11.3 85.6 ± 12.5 <0.05Estrus duration (h) 11.0 ± 5.3 13.7 ± 5.9 nsc

Interval beginning of estrus to ovulation (h) 29.3 ± 4.9 31.6 ± 4.4 nsInterval end of estrus to ovulation (h) 17.7 ± 3.7 18.4 ± 5.1 nsNumber of mounts per estrus 23.0 ± 15.6 28.2 ± 23.2 nsDuration per mount (s) 3.7 ± 0.7 3.5 ± 0.8 nsTotal duration of mounts per estrus (s) 89.9 ± 75.3 103.7 ± 93.0 nsMaximum � of ovulatory follicle (mm) 13.7 ± 1.4 13.5 ± 0.7 nsP4 at PGF2� (ng/mL) 2.79 ± 0.98 4.85 ± 1.26 <0.05P4 at synchronized estrus, D0 (ng/mL) 0.20 ± 0.08 0.33 ± 0.08 nsP4 on D5 (ng/mL) 1.50 ± 0.40 1.27 ± 0.44 ns

pm

rPoptesfdd(s

3

lcohtbrstp

PibaaaspFade

a Early luteal phase.b Late luteal phase.c No significant difference; P4, progesterone.

ercentage of estruses detected that were true estruses (aeasure of the false positives; Xu et al., 1998).The present study was performed using a completely

andomized design with two treatments, corresponding toGF2� administration during the early or late luteal phasef the estrous cycle. The data were analyzed using the SASrogram (Statistical Analysis System Institute Inc.). Con-inuous variables were previously tested for normality andquality of variances and some data were log [Log(X + 1)] orquare-root [SQRT(X + 1/2)] transformed. Original or trans-ormed data were then analyzed using t tests. Frequencyata (binary responses) were analyzed by the �2 test. Theata were reported as the means ± standard deviationsSD) and differences with P-values ≤ 0.05 were consideredignificant.

. Results

Following PGF2� administration during the early or lateuteal phase, the radiotelemetry performed at 90% effi-iency (45/50 detected in estrus) and 100% accuracy (45/45vulations). There was no mounting behavior in the fiveeifers whose estrus was not detected by radioteleme-ry, although these heifers had silent estrus, as confirmedy ovulation (10%; n = 5/50 not detected in estrus). Theadiotelemetry efficiency for the detection of estrus wasimilar (P > 0.05) for PGF2� treatment provided duringhe early (90.9%; 30/33) or late (88.2%; 15/17) lutealhase.

The means of various estrous characteristics followingGF2� administration during the early or late luteal phasen buffalo heifers (n = 50) and the distributions for theeginning of estrus and ovulation are described in Table 1nd Fig. 1, respectively. The intervals between PGF2�

dministration and the beginning of synchronized estrusnd between PGF2� administration and ovulation werehorter (P < 0.05) for heifers treated during the early lutealhase compared to the late luteal phase (Table 1 and

ig. 1). However, there was no impact (P > 0.05) of PGF2�

dministration during the early or late luteal phase on theuration of estrus, the interval between the beginning ofstrus to ovulation, the interval between the end of estrus

to ovulation and the maximum diameter of the ovulatoryfollicle (Table 1).

Furthermore, PGF2� administration during either theearly or late luteal phase had no impact (P > 0.05) on thenumber of mounts per estrus, the duration of mounts, thetotal duration of mounts per estrus (Table 1) and the distri-bution of mounts during the day (morning, 27.1 vs. 23.5%;afternoon, 22.6 vs. 19.2%; night, 23.8 vs. 26.3%; and dawn,26.5 vs. 30.9% for the early or late luteal phase, respec-tively). Additionally, the intensity and duration of detectedestrus was not influenced (P > 0.05) by the day of PGF2�

administration. Approximately 75.6% of estruses had a highintensity, with 57.8% having a long duration and 17.8% hav-ing a short duration. The remaining 24.4% of estruses had alow intensity, with 22.2% having a long duration and 2.2%having a short duration.

On the day of the second PGF2� administration, plasmaprogesterone concentrations were low for heifers treatedduring the early luteal phase compared to the late lutealphase (P < 0.05, Table 1). Thereafter, PGF2� administrationcaused 100% luteolysis (plasma progesterone < 1 ng/mLwithin 48 h of administration) regardless of the luteal phaseon the day of administration. Similar concentrations ofplasma progesterone (P > 0.05) were recorded on the dayof synchronized estrus (D0) and on day 5 after estrus inheifers treated with PGF2� during the early or late lutealphase (Table 1).

4. Discussion

In this pioneering study, the use of radiotelemetryexhibited an excellent efficiency and accuracy for thedetection of estrus in buffalo heifers. Using the samemethodology for estrous detection, a similar efficiency andaccuracy was reported in beef (Stevenson et al., 1996) anddairy cattle (Palmer et al., 2010; Xu et al., 1998). In thisstudy, only 10% of buffalo heifers exhibited silent estruscompared to other reports for this species, which varying

from 14.3% (Jha, 2011) to 62.5% (Seren and Parmeggiani,1997) using the traditional method of visual estrous detec-tion. In dairy cattle, 17% were reported to exhibit silentestrus using the HeatWatch® system (Palmer et al., 2010).

R.M. Porto-Filho et al. / Animal Reproduction Science 144 (2014) 90– 94 93

(A)

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48 -| 60 60 -| 72 72 -| 84 84 -| 96 96 -| 108 108 -| 12 0

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)

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earl y lut eal phas e

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Beg

inni

ng o

f es

trus

(%)

Hours after PG

early luteal phase

late luteal phase

PGF2� a

Fig. 1. Distribution of beginning of estrus (A) and ovulation (B) followingheifers.

The response of buffalo heifers to a luteolytic dose ofPGF2� was satisfactory because 90% of the heifers exhib-ited estrus following luteolysis. In previous studies, similartreatments exhibited estrous behavior in 87–90% of buf-faloes (Baruselli et al., 2003; Brosrekar et al., 1994; Diazet al., 1994). Moreover, following PGF2� administrationduring the early luteal phase, the shorter interval to syn-chronized estrus and to ovulation, compared to PGF2�

administration during the late luteal phase, was alsoobserved in dairy (Kastelic et al., 1990) and beef cattle (Saleset al., 2007). This could be because, at the time of luteolysisduring the early luteal phase, the dominant follicle was in adevelopmental stage and ovulated, whereas, at the time ofluteolysis during the late luteal phase, the dominant folliclewas in a static phase and failed to ovulate. In the latter case,the dominant follicle from the subsequent follicular wavemight have ovulated, resulting in a longer interval betweenPGF2� administration and the manifestation of estrus orovulation (Kastelic et al., 1990).

The duration of recorded estrus (11.8 ± 5.6 h) was con-siderably shorter compared to reports in buffaloes wherevisual detection of estrus was performed (20.5 ± 1.2 h,Kanai and Shimizu, 1986; 28.3 ± 21.3 h, Moioli et al., 1998;

dministration during early or late luteal phase of estrous cycle in buffalo

23.8 ± 8.2 h, Vale, 1983 and Vale and Ribeiro, 2005). Thesedifferences may be attributed to the improved precisionand accuracy for detecting the true beginning and end ofestrous behavior when using an electronic detection sys-tem compared to a visual one.

In this study, the timing of PGF2� administration duringthe luteal phase had no impact on the intervals betweenthe beginning or end of estrus and ovulation, the number ofmounts per estrus, the duration of mounts, the total dura-tion of mounts per estrus and the estrus intensity. Theseobservations suggest that, regardless of the luteal phase inwhich buffalo heifers receive PGF2�, all heifers in estruswill have similar estrous behaviors and ovulatory follicledevelopment. Studies are not available in buffalo or cattlefor comparison with these results.

Furthermore, the distribution of mounts throughoutthe 24 h day was uniform. This is in contrast to previ-ous reports in buffaloes that demonstrated more intensereproductive behavior being exhibited at night during the

summer/fall in a warm Amazonia region (Vale et al., 1984).This could be due to the differences in climate and season(fall/winter) between these studies. However, the distri-bution of mounts in beef (Stevenson et al., 1998) and dairy

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4 R.M. Porto-Filho et al. / Animal R

ows (Dransfield et al., 1998; Xu et al., 1998) throughout aay that was broken into four periods of six hours each wasimilar to that of the buffalo heifers presenting this study.

Plasma progesterone concentrations differed betweenuffaloes receiving PGF2� during the early and late lutealhases. Lower plasma progesterone in heifers treated dur-

ng the early luteal phase can be associated with theaturity of the CL during the estrous cycle (Baruselli et al.,

997b; Ohtani et al., 1998; Perez-Marin, 2009; Sartorit al., 2004). However, plasma progesterone concentra-ions on the day of synchronized estrus (D0) and onay 5 after estrus were similar between heifers treateduring the early or late luteal phase. This suggests thathe day of PGF2� treatment during the luteal phase haso impact on luteal activity subsequent to synchronizedstrus.

In conclusion, radiotelemetry is a highly efficient andccurate system for estrous detection in buffalo heifers.dministration of PGF2� during the early luteal phaseesults in shorter intervals to the beginning of estrus andvulation. However, luteolytic treatment during the earlys. the late luteal phase causes no significant differencesith regard to the exhibition of estrous behavior, the max-

mum diameter of the ovulatory follicle and the subsequentuteal function.

cknowledgements

Conselho Nacional de Desenvolvimento Científico e Tec-ológico (CNPq).

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