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Cuban Journal of Agricultural Science

Print version ISSN 0864-0408On-line version ISSN 2079-3480

Cuban J. Agric. Sci. vol.50 no.1 Mayabeque Jan.-Mar. 2016


Cuban Journal of Agricultural Science, 50(1): 61-69, 2016, ISSN: 2079-3480




Clinical characteristics of induced estrus with hormones, and its relation with gestation percentage in nulliparous female water buffaloes


Características clínicas del estro inducido con hormonas y su relación con el porcentaje de gestación en búfalas de río nulíparas



C. Gallego,I Eunice Oba,IV J.C. Alonso,II L.M. Fraga,I R. García López,I Mildred Méndez,I E. Padrón,III

IInstituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba.
IIUniversidad Agraria de La Habana “Fructuoso Rodríguez Pérez”, Carretera de Tapaste y Autopista Nacional, San José de las Lajas, Mayabeque, Cuba.
IIIEmpresa Genética Pecuaria “El Cangre”, Mayabeque, Cuba.
IVUniversidad Estatal Paulista, Campo de Botucatú, San Paulo, Brasil.




An amount of 82 nulliparous female water buffaloes were studied to compare the signs of induced estrus with hormone protocols (P). Treatments PI (n=29), PII (n=28) and PIII (n=25) received 2.0 mg of estradiol benzoate and intravaginal device of progesterone for nine days. After removing the device, 0.5 mg of prostaglandin F, 500 IU of equine chorionic gonadotropin and 0.5 mg of estradiol cypionate (PI and PII) were applied. In PIII, cypionate was replaced by 1000 IU of human chorionic gonadotropin after 48 h. Inseminations were carried out between 48-50 (PI), 56- 58 (PII) and 14-16 h (PIII) after the last hormone. A generalized linear mixed model was applied, which considered the protocol as fixed effect, and the animal nested in the protocol as random effect. Tukey-Kramer test was applied for the multiple comparisons. The animals of PI showed higher swollen vulva (77.5 %) and higher pink coloration of the vaginal vestibule (75.2 %) than those from PII (16.7 and 16.26 %) and PIII (15.4 and 13.26%). Female buffaloes of PI (61.6 %) and PII (81.2 %) showed higher secretions than those of PIII (22.8 %). Uterine texcitement, tone cervical passage and insemination time showed no differences among protocols. Cervical passage showed a positive and high correlation with the gestation percentage. This last was negatively related to excitement and time during the insemination. It can be concluded that differences in the expression of signs had no influence on the percentage of gestation among protocols.

Key words: female buffaloes, clinical signs, estrus, hormonal protocol.


Se estudiaron 82 búfalas nulíparas para comparar los signos del estro inducido con tres protocolos hormonales (P). Los P I (n=29), P II (n=28) y P III (n=25) recibieron 2.0 mg de benzoato de estradiol y un dispositivo intravaginal de progesterona durante nueve días. Al retirarse el dispositivo, se aplicó 0.5 mg de prostaglandina F, 500 UI de gonadotropina coriónica equina y 0.5 mg de cipionato de estradiol (PI y PII). En el PIII se sustituyó el cipionato por 1000 UI de gonadotropina coriónica humana 48 h posteriores. Las inseminaciones se realizaron entre 48-50 (PI), 56-58 (PII) y 14-16 h (PIII) después de la última hormona. Se aplicó un modelo lineal generalizado mixto, que consideró al protocolo como efecto fijo y como aleatorio, al animal anidado en el protocolo. Se aplicó dócima de Tukey-Kramer para la comparación múltiple de medias. Los animales del PI mostraron mayor edema vulvar (77.5 %) y coloración rosada del vestíbulo vaginal (75.2 %) que los del PII (16.7 y 16.26 %) y PIII (15.4 y 13.26%). Las búfalas de los PI (61.6 %) y PII (81.2 %) mostraron mayores secreciones que en el PIII (22.8 %).La turgencia uterina, excitemento, permeabilidad cervical y tiempo de inseminación no mostraron diferencias entre los protocolos. La permeabilidad cervical mostró correlación positiva y alta con el porcentaje de gestación. Este último se relacionó negativamente con el excitemento y tiempo durante la inseminación. Se concluye que las diferencias en la expresión de los signos no influyeron en el porcentaje de gestación entre los protocolos.

Palabras clave: búfalas, signos clínicos, estro, protocolo hormonal.




The use of artificial insemination (AI) in genetic programs of Bufalipso in Cuba is not in the same proportion than bovine cattle, which leads to a delay in the genetic process and the desired productive levels (Fraga et al. 2013). The main causes that limit the extensive use of AI in female buffaloes is the heat detection and the most appropriate moment to carry it out, because the clinical signs of estrus are less evident (Rossi et al. 2014). 

Several therapies of estrus synchronization are developed for the increase of fertility (Mohan and Prakash 2010). The most practiced ones are those that synchronize the wave of follicular development, estrus and ovulation, so animals can be inseminated at fixed time (FTAI). Nevertheless, it is necessary to contribute to the knowledge of endocrine and ethologic factors that take part in the period of sexual behaviour, in order to develop strategies to improve fertility of species (Porto Filho et al. 2014).

Occurrence, intensity and duration of provoked estrus may vary according to the different hormonal therapies. Mohan and Prakash (2010) stated that this performance may contribute to efficiency of hormonal treatments from the response of reproductive organs, as consequences of hormonal action. Quirino et al. (2013) and Mirmahmoudi et al. (2014) studied the expression of several clinical symptoms and signs of natural and induced estrus. However, there are no studies that refer the performance of estrus during the FTAI with the use of different hormonal protocols.

The objective of this study was to compare the percentage of expression of clinical signs of estrus and other aspects during the FTAI, and its relation to gestation percentage, with the use of different hormonal protocols that contain steroidal hormones (progesterone and estrogens) prostaglandins F, human (hCG) and equine chorionic gonadotropin (eCG) during the period of low breeding season.



Stage of study, localization and selection of animals. This research was carried out in July, in a development unit belonging to Empresa Pecuaria Genética “El Cangre” (Latitude 23º 5.35’ North; Longitude 82º 30.5’ West), located in Güines, Mayabeque province, Cuba.

An amount of 82 nulliparous female water buffaloes with 23 months old, 378.62 ± 23.78 kg of liveweight and 3.0 points of body condition. These animals showed no gynecological anomalies at transrectal palpitation and were free of infect-contagious diseases (brucellosis and tuberculosis).

Management and feeding. During daytime, animals had access to a continuous grazing (06:00 am up to 07:00 pm), with ponds and natural shadow. At night, they remained stabulated and water at will. Star grass (Cynodon nlemfuensis) prevailed as base feed, without fertilization or irrigation, and, in lower proportion, some varieties like hurricane grass (Botriochloa pertusa) and bahia grass (Paspalum notatum). Grasses under those conditions showed an availability of 37.0 kg of DM. female buffalo-1 during rainy season, which allowed to cover the requirements on a dry basis.     

Experimental procedure. Three groups of female buffaloes were created, which represented the evaluated protocols (P). The animals of PI (n=29), PII (n=28) and PIII (n=25) received 2.0 mg of estradiol benzoate (BE2, Ourofino®) intramuscularly (IM) and a progesterone-releasing intravaginal device (PRID of P4, Tecnopec®) for nine days. Female buffaloes of PI and PII received 0.5 mg of prostaglandin F (PGF, Ciosin®), 500 international units (IU) of equine chorionic gonadotropin (eCG, Novormon®) and 0.5 mg estradiol cypionate (EC2, E.C.P®) (IM), after withdrawing the ID of P4. Animals of PIII received the same hormonal treatment, except the CE2. At 48 h after the treatment, 1000 IU of human chorionic gonadotropin (hCG, Chorulon®) were applied (Figure 1).

FTAI was conducted between 48-50 h in PI (08:00 am), 56-58 h in PII (04:00 pm) and 14-16 h in PIII (08:00 am), after the last hormonal application. At the time of the FTAI, signs of estrus (swollen vulva, vaginal vestibule coloration, mucus discharge cervico-uterine tone, and color of uterine horns) and other aspects during insemination (excitement, cervical passage and time of FTAI ≤ 60 s) were evaluated, with the purpose of comparing the expression percentage among hormonal protocols. Pregnancy diagnosis was performed at 45 d through the rectum (manual and ultrasound).

Statistical analysis. Data were processed using a generalized linear mixed model (Wolfinger and O'Connell 1993), using the PROC GLIMMIX of SAS (2010) version 9.3. All variables were considered as binomial (1 presence and absence 0) and the model took into account a binomial distribution and the logit was considered as bond function, offering the retransformed values. The treatment (hormonal protocol: three levels) was considered as fixed effect and the animals (25-29) nested in this treatment were used as random effect. Covariables of liveweight (LW) and age at the moment of FTAI were evaluated, which were not considered in the final model because they were not significant.

The test of Tukey, modified by Kramer (1956) for multiple comparison of means, with significance level of P <0.05, was applied. By means of PROC MEANS of SAS (2010), position and dispersion statistics (mean, standard deviation, coefficient of variation, minimum and maximum values) were determined to binomial variables that showed no differences among hormonal protocols, in order to know their general performance. The CORR procedure of SAS (2013) program were used to estimate Pearson correlations among the variables studied for each hormonal protocol.

The following model was used:

Yjk= μ+ Aj+ Cj(Ak) +ejk


Yjk= f(µ) expected phenotypic value of studied variables, according to the function of specific bond

μ = mean or intercept

Aj  = fixed effect of the  j-th hormonal protocol (k= I, II, III)

Ck (Aj) = random effecto of the k-th female buffalo nested in the j-th hormonal protocolo

ejk= random error due to each observation NID~(0, s2e)



Swollen vulva, dark pink coloration of vaginal vestibule and the presence of mucus discharge cervico-uterine at transrectal palpation were signs that showed differences among hormonal protocols (Table 1), while the uterine tone, nervous excitement, time of FTAI ≤ 60 s and cervical passage showed no significant effect.

Percentages and differences between treatments suggest that heat of animals from protocol I had better expression, although it had no influence on pregnancy percentage (figure 2). Results could be related to an increase of estrogen concentration in blood of animals and, consequently, a superior expression of heat performance during the period of the observation. In general, during other hours after the application or not of CE2 (II and III), the signs occurred in a reduced percentage of animals. This performance could be caused by the time since the implementation of CE2 until the observation during the FTAI. This period of time was shorter (PI = 48-50 h) with respect to the other hormonal protocols (PII = 56-58 h and PIII = 62-64 h).

These results are based on evidence from studies of Mohan et al. (2009), but in lactating animals. These authors found that 100% of female buffaloes showed the highest concentrations of estradiol in the blood during the first 24 h (range 12-26 h) after application of BE2, with a tendency to gradually decrease.

Similar results reported Mohan and Prakash (2010), who also observed that, in 60% of animals of the same productive category, there was a period of time from the treatment at the beginning of swollen vulva with a range between 18.2 ± 1.01 and 40.0 ± 1.85 h, using the Heatsynch® protocol. Hiremath (2013) reported that 67.8% of female buffaloes in milk production showed equal clinical signs between 36 and 48 h, after using BE2, 24 h after the removal of PRIDP4.

Research conducted by Kajaysri et al. (2015) also demonstrated increased expression of clinical signs of estrus between 32 and 36 h after removal of the release device of P4 (CIDR®).

Similar performances were reported by Pancarci et al. (2002) and Mohan and Prakash (2010), who found 86 and 60 % of female buffaloes with visible secretions, when using CE2 and BE2 as ovulation synchronizer, respectively. These authors described a range of appearance between 22 and 44 h.

This study showed that swollen vulva and secretions remained during a slightly superior period, which could be caused by the frequency and experience of estrus detection, by the nutritional state of animals, dosage and type of hormone. CE2, the estradiol ester used in PI and PII, has an average life in blood superior to BE2. This effect could explain most of the permanence of signs observed in this research. The study method of heat also influenced on the results because the observation was only performed during the FTAI. Manipulation and stimulation of the insemination catheter in genital organs could increase uterine contractions that stimulate the fluid evacuation.

Other possible differences may be related to productive and reproductive category because most of the previous studies were performed in milk-producer multiparous female buffaloes. Sartori and Mendes (2010) stated that cows, during lactation, show a period of lower estrus, due to a decrease of estradiol concentrations circulating in blood, as a consequence of a more accelerated hepatic metabolism.

The uterine tone and cervical passage showed a high percentage (75-79 and 80-86 %, respectively) in all hormonal protocols (table 2). Mohan et al. (2009) pointed out, as a distinct characteristic, the strong increase of uterine tone and cervical dilatation during natural or induced estrus, apart from the used hormones. However, Atanasov et al. (2011) found no differences in uterine tone when the animals presented follicules with lower or higher diameter than 10.0 mm-1. Studies conducted by Martin et al. (2009) and Lima et al. (2011)  confirm the high sensitivity of the uterus to the effect of estradiol, due to the high number of estrogen receptors, more than in other segments of the genital tract.

The action of the eCG stimulating follicle could be the cause of not having differences in the turgidity of uterine horns and cervical passage among protocols because this hormone provokes an increase in the development of dominating follicles and, consequently, an increase of estrogen concentration in blood of follicular origin. The nervous excitement of animals showed a similar performance in all protocols, which could justify the insemination of only between 30 and 37 % of female buffaloes during a time ≤ 60 s (table 2).

The time during the AI and excitement showed a negative and mean correlation with gestation in all hormonal protocols (table 3), which could have an influence on the act of insemination technique and, consequently, on fertility of animals. Nervous excitement of animals could be related to management and scarce daily contact with humans (without milking). This performance was similar to that reported by Tito et al. (2010), who stated that young animals may be stressed faster. According to these authors, frequent moving, attempts to go back, muscular contractions, defecation and jumps, at the moment of FTAI, may have a negative influence on the skills of the technician and, consequently, on the insemination time. 

Vendruscolo et al. (2005) demonstrated that the speed of FTAI has a significant influence on gestation percentage, and it is related to cervical passage and excitement of animals. The statistical analysis of that study evidenced that probability of animals to gestate, from the AI performed before two minutes, was 3.45 times higher than when the time was superior.

Cervical passage and gestation also showed high and positive relation (table 3). Not exposing the spermatozoids to defense cells (neutrophils and lymphocytes) located in the cervix, advancing the sperm route and locating the semen in areas of the uterine area, which contain higher concentration of substances that stimulate the acrosome reaction and neutralize handicapping factors of seminal plasma, could avoid the decrease of its concentration, the energy waste and increase of fertilizing ability (Vecchio et al.  2013).

It can be concluded that there are differences in the expression of clinical signs of estrus (swollen vulva, dark pink coloration of the vaginal vestibule and cervical and uterine secretions), according to the used hormonal protocols, although they had no influence on gestation percentage. Fertility of animals is favored by cervical passage. However, it is affected with the increase of excitement and the time of FTAI is extended, which are practical aspects to be considered in order to improve reproductive indexes for future insemination programs.



Thanks to the Coordinación de Perfeccionamiento para la Educación Superior de la República de Brasil (CAPES) for the financing (Project 96/2010) and to the Empresa Genética Pecuaria “El Cangre” for facilitating the implementation of this study.



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Received: February 26, 2015
Accepted: March 30, 2016



C. Gallego, Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba. Email:

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