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Cuban Journal of Agricultural Science

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

Cuban J. Agric. Sci. vol.49 no.3 Mayabeque July.-Sept. 2015




Pregnancy rate obtained with four hormonal protocols for fixed-time artificial insemination in nulliparous water buffaloes (Bubalus bubalis)


Tasa de gestación obtenida con cuatro protocolos hormonales para la inseminación artificial a tiempo fijo en búfalas de río nulíparas (Bubalus bubalis)



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

IInstituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba.
IIUniversidad Agraria de La Habana, Carretera de Tapaste y Autopista Nacional, San José de las Lajas, Mayabeque, Cuba.
IIIEmpresa Pecuaria Genética Bubalina «El Cangre», El Cangre km 6 1/2, Güines, Mayabeque, Cuba.

IVUniversidad Estatal Paulista. Campus de Botucatu, Estado de São Paulo, Brasil.




A total of 120 nulliparous water buffaloes were used to evaluate the effect of different hormonal protocols with fixed-time artificial insemination on the pregnancy rate. Protocol 1 received 2.0 mg of estradiol benzoate and a progesterone intravaginal device for nine days. After the device removal, 0.5 mg of Prostaglandin F2α, 500 UI of equine chorionic gonadotropin and 0.5 mg of estradiol cypionate were applied. Protocol 2 was similar to 1, but without the chorionic gonadotrophin. In protocol 3, two doses of 0.5 mg of Prostaglandin F2α were injected, with an interval of 11d, and 0.5 mg of estradiol cypionate was added to the last dose. Protocol 4 was similar to 3, but, after 48h of the last Prostaglandin F2α, 0.01 mg of Buserelin Acetate was injected. Time-fixed artificial inseminations were performed between 56 and 58 h (protocols 1, 2 and 3) and between 20 and 24 h (protocol 4) after the last hormonal application. Pregnancy rates in protocol 1 (60.8%), 2 (50. 3 %) and 4 (57.3 %) were superior (P<0.05) to those of protocol 3 (16.0 %). The equine chorionic gonadotrophin had no influence on the increase or decrease of fertility. In the protocol with double doses of Prostaglandin F2α and estradiol cypionate, fertility decreased, but increased when the estradiol was substituted by Buserelin Acetate. It can be concluded that protocols 1, 2 and 3 synchronize the estrum and ovulation with good pregnancy rates, and it is possible to apply fixed-time artificial insemination for increasing birth rate in herds and achieving economic benefits.

Key words: hormonal protocols, fixed-time artificial insemination, nulliparous buffaloes.


Se utilizaron 120 búfalas de río nulíparas para evaluar el efecto en la tasa de gestación de diferentes protocolos hormonales con inseminación artificial a tiempo fijo. El protocolo 1 recibió 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 F2α, 500 UI de Gonadotropina coriónica equina y 0.5 mg de Cipionato de estradiol. El protocolo 2 fue similar al 1, pero sin la Gonadotropina coriónica. En el protocolo 3, se inyectaron dos dosis de 0.5 mg de Prostaglandina F2α, con intervalo de 11 d, conjuntamente con la última se administró 0.5 mg de Cipionato de estradiol. El protocolo 4 fue similar al 3, pero luego de 48 h de la última Prostaglandina F2α, se inyectó 0.01 mg de Acetato de Buserelina. Las inseminaciones artificiales a tiempo fijo se realizaron entre 56 y 58 h (protocolos 1, 2, 3) y 20-24 h (protocolo 4) posteriores a la última aplicación hormonal. Las tasas de gestación en el protocolo 1 (60.8%), 2 (50. 3 %) y 4 (57.3 %) fueron superiores (P < 0.05) a la del protocolo 3 (16.0 %). Se comprobó que la Gonadotropina coriónica equina no influyó en el incremento o reducción de la fertilidad. En el protocolo con doble dosis de Prostaglandina F2α y Cipionato de estradiol, la fertilidad disminuyó, y aumentó cuando se sustituyó el estradiol por Acetato de Buserelina. Se concluye que los protocolos 1,2 y 3 sincronizan el estro y la ovulación con buenas tasas de gestación, y que es posible aplicar la inseminación artificial a tiempo fijo para incrementar la natalidad en los rebaños y lograr beneficios económicos.

Palabras clave: protocolos hormonales, inseminación artificial a tiempo fijo, búfalas nulíparas.




Artificial insemination in buffaloes, like in other domestic species, contributes to health improvement and productive progress of herds (Hafez, 1995). However, in order to achieve this purpose, female buffaloes face different difficulties like poor performance and expression of heat, and variability of its appearance during the day. This makes difficult the notice of the estrus and the determination of the most adequate moment to perform a successful insemination (Das and Kan 2010). In addition, there are other limitations related to the short period of service acceptance, variability of ovulation time, and ovarian inactivity during the summer months (El-Wishy 2007).         

In order to solve these problems, Mishra et al. (2007) stated the use of estrus induction and synchronization methods with hormonal protocols (progesterone, estrogens, prostaglandins and gonadotropins) applied to female cattle, and obtain good results in fertility. Nevertheless, as these treatments also need to detect the heat, they do not solve the problems in this species because they do not guarantee a proper synchronization between the estrus and the ovulation (Mohan and Prakash 2010). Therefore, nowadays, protocols that also include the synchronization of ovulation for performing the fixed-time artificial insemination (FTAI) are recommended (Porto Filho et al. 2014).

The objective of this paper was to evaluate the effect of four hormonal protocols, which would induce the estrus and synchronize ovulation, on pregnancy percentage and expenses of the FTAI of nulliparous female water buffaloes during the season of higher estrus cyclicity.



Stage of study, location and selection of animals. The research was carried out on October, 2013, in a development unit belonging to the Empresa Pecuaria “El Cangre” (latitude 23º 5.35’ N; longitude 82º 30.5’ W), in Güines, Mayabeque province, Cuba. An amount of 120 nulliparous female water buffaloes were used, with ages between 22 and 24 months old and mean live weight of 374.97 ±21.94 kg, with no gynecological anomalies at rectal palpation, and free of diseases like tuberculosis and brucellosis.  Before applying the hormonal protocols, an examination of internal genital organs was performed (rectal palpation and ultrasonography) to 30 % of the animals in each group, with the purpose of verifying the ovarian cyclicity.

Management and feeding. Female buffaloes were managed through day and continuous grazing (from 06:00 a.m. to 07:00 p.m.) with access to lakes and natural shade. At night, they stayed remained stabulated, without supplements and complements, and received water at will. Star grass (Cynodon nlemfuensis), without fertilization and irrigation, prevailed as base food. There were also natural grasses included, mainly Bothriochloa pertusa and Paspalum notatum. Under these conditions, grasses presented an availability of 37.00 kg of
DM/female buffalo and covered all the requirements for female buffaloes.

Experimental design. Four groups of female buffaloes (n=30/group) were established, which presented a body condition superior to 3.0, in a scale between 1 and 5 points (Kapa and Alapati 2013). Four hormonal protocols (P) were applied to these groups. P1 received 2.0 mg of estradiol benzoate (BE2, Sincrodiol®) and a progesterone intravaginal device (DIVP4, Primer®) for nine days. After the device removal, 0.5 mg of Prostaglandin F2α (PGF2α, Ciosin®), 500 UI of equine chorionic gonadotropin (eCG, Novormon®) and 0.5 mg of estradiol cypionate (CE, E.C.P®) were applied. P2 was similar to P1, but without the eCG. In P3, two doses of 0.5 mg of PGF2α were injected, with an interval of 11d, and 0.5 mg of CE was added to the last dose. P4 was similar to P3, but, after 48h of the last PGF2α, 0.01 mg of Buserelin Acetate (GnRH, Sincroforte®) was injected. Time-fixed artificial inseminations were performed between 56 and 58 h (P1, P2 and P3) and between 20 and 24 h (P4) after the last hormonal application. All the hormones used, except DIVP4, were administered intramuscularly. Figure 1 shows the diagrams of the treatments proposed for TFAI.

Pregnancy diagnosis was carried out at 45 d, using a transrectal ecography (AQUILA PRO Esaote Pie Medical®), with a linear probe of 8.0 and 10.0 MHz. The TFAI was performed with semen from Mediterranean breed, with fertility certification, from Italy (Central de Inseminación Artificial “Coffa Semen Bubalino”). For its preservation, it was stored in 0.50 mL straws and in tanks of liquid nitrogen (-196 °C). The semen defrosted at 37.0 C° for 45 seconds, previous to the insemination with a Cassou insemination gun.

Statistical analysis. Mixed generalized linear models were applied (Wolfinger and O’Connell 1993), with help of GLIMMIX from SAS (2013), version 9.3. The model considered the treatment (hormonal protocol) as fixed effect, and the animal nested in the treatment as a random effect. The co-variable of liveweight at TFAI was analyzed, but it was not significant so, it was not included on the final model. In order to determine the differences among pregnancy rates obtained in each hormonal protocol, the test of Tukey, modified by Kramer (1956), was used, with a signification level for P<0.05. The pregnancy variable was considered as binomial, and its analysis was contemplated in the program (binomial distribution and logit link). The values were retransformed:

Used model

 Yjk  = μ+ Aj+ Cj (Ak) +  ejk


Yjk = f (µ) fenotypical value expected from the studied variables, according to its specific function of link

μ = mean or intercept

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

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

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

Expenses of hormonal therapies. The expenses were determined from the cost of each hormone (Brazil) and the expenses for each hormonal protocol, taking into account the hormone and the dose used. Expenses of hormonal protocols (USD) were calculated per animal and per pregnancy. Pregnancy expenses (GG) per each hormonal protocol was obtained through the following expression:



GTP= total expenses per each protocol per group

NBP= number of pregnant female buffaloes in each group



Regarding the variable pregnancy percentage, the analysis of variance showed significant effect (P < 0.05) among the hormonal treatments during the season of larger reproductive activity. The combination of two doses of PGF2α, with an interval of 11 d, and the administration of CE (P3) showed a pregnancy percentage significantly inferior regarding the rest of the hormonal protocols (1, 2, 4), which had no differences among them. After analyzing the results obtained between treatments 1 and 2, it was confirmed that removing eCG (P2) did not affect the pregnancy percentage. However, the pregnancy percentage increased significantly in the protocol that substituted CE for GnRH (P4), similar to P1 and P2. These results demonstrate that CE affected fertility in the treatments with double doses of PGF2α (figure 2).

Crudelli et al. (2005) reported similar results to those reached in P3, after finding pregnancy percentages of 11.10 % in primiparous buffaloes with continuous lactation. The second application of GnRH of Ovsynch® protocol was replaced by the administration of CE the same day in which PGF2α was applied. The cited authors considered that low pregnancy indexes could be caused by an anticipated appearance of ovulation regarding the time of FTAI (58-60 hr), because after 48h of the application of CE, follicles with ovulation capacity (follicular diameter of 8.00 and 8.50 mm) were found.

Although this study did not determine the follicular development during the hormonal treatment, the results could indicate that CE stimulated earlier the ovulation, regarding the time of FTAI (56-58 hr) in P3. This situation was similar to that observed by other researchers. It is also possible that nulliparous female buffaloes, during the stage of higher ovarian activity, presented, in advance, follicles with ovulatory capacity (<48h). This analysis relates to studies by Presicce et al. (2005), who confirmed that nulliparous female buffaloes showed higher daily follicular growth rate (1.30 mm.d-1) than primiparous females (0.95 mm.d-1).

De Rensis and Lopez-Gatius (2007), in a similar season, also obtained inferior values to those of this study (P1, 2 and 4) in female buffaloes under lactation. This disagreement could be related to the body and reproductive state of animals at the beginning of the postpartum hormonal treatment, as a result of the negative energy balance they experience during lactation, which interferes on the range and frequency of FSH (Follicle-Stimulating Hormone) pulses and the pre-ovulatory peak of LH (Luteinizing Hormone), and, consequently, the post-partum ovarian activity.

Animals that received double doses of PGF2α and GnRH showed a favorable pregnancy percentage for the species, which indicated an effective synchronization between the ovulatory period and the moment of the fixed-time artificial insemination. This therapy could represent an interesting alternative in the programs of FTAI in nulliparous buffaloes during the season of higher ovarian activity.

Results confirm the criterion of Ohashi (2003), who stated that therapies combining PGF2α and GnRH are relatively easy because they only imply the parenteral supply of products. In addition, its current costs are affordable, which is an important aspect for a wider use of programs of FTAI in buffaloes.

 Marques et al. (2005) state the positive effect of eCG on the increase of ovulatory follicle size, which improves the ovulation and pregnancy indexes. However, Baruselli and Carvalho (2005) pointed out that buffaloes show efficient responses with hormonal protocols for TFAI without eCG during the favorable reproductive season. Nevertheless, during the unfavorable reproductive season, hormonal therapies need eCG to stimulate the follicular development.

The criteria of these authors agree with the results of this study, in which the animals from P1 and P2 showed similar pregnancy percentage (figure 2). This study demonstrated that the eCG can be excluded, during the season of more ovarian activity, from the protocols using DIVP4, associated to estradiol esters, luteollitic and synchronizing ovulation products, with the objective of stimulating and normalizing the follicular wave, the estrum and ovulation. 

As table 1 shows, the protocols that did not use eCG (P2, P3 and P4) as stimulant of follicular development showed the lowest expenses per animal, regarding the group that included this hormone (P1). Similar results were found by Noguera et al. (2013), after comparing the expenses of Ovsynch® (9.50 USD) and Cl-Synch (19.33 USD) therapies with a protocol that used and intravaginal sponge of synthetic P4 (Pregna-Heat-E®, VIATECA), BE2 (2,0mg), eCG  (400 IU, Folligon®) and GnRH (0.0084 mg Buserelin Acetate, Conceptal®) in nulliparous female buffaloes during the season of higher ovarian activity (43.13 USD), without finding differences regarding the pregnancy percentage at fixed-time artificial insemination. However, the lowest expenses per gestation were shown by the protocol that associated PGF2alpha and GnRH (PIV) hormones.

Unlike bovines, most of hormonal therapies in buffaloes synchronize the wave of follicular development and estrum, and ovulation, due to the variations in process duration. In this sense, other input are needed, which make therapies more expensive and limit technology implementation. However, during the favorable season for reproduction, expenses can decrease because it is possible to use protocols that do not use hormones (eCG) to stimulate the follicular development (Baruselli and Carvalho 2005).

It was demonstrated that pregnancy rates in P1 (60.8 %), P2 (50.3 %) and P4 (57.3 %) were superior (P < 0.05) to P3 (16.0 %). There was no influence of eCG on the increase or reduction of fertility. In the protocol with double doses of PGF2α and CE, fertility decreases, and it increases when the estradiol is substituted by GnRH. It was evident that the protocol with the most reduced cost (P3) had lower percentage and higher expenses per gestation, regarding the rest of the therapies. However, P1 and P2 showed a fertility that did not differ from P4, although with expenses to obtain a gestation superior to this last group (table 1), which is an aspect that classified it as the most efficient treatment. It can be concluded that protocols 1, 2 and 3 synchronize with the estrum and ovulation with a good pregnancy percentage. It is also possible to apply the FTAI for increasing birth rate in buffalo herds, with economical benefits. 



Thanks to the Coordinación de Perfeccionamiento para la Educación Superior of the   Brazil Republic (CAPES) for the financing (Project 96/2010) to perform this study.



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Received: September 25, 2014
Accepted: April 1, 2015



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

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