Livestock Research for Rural Development 25 (10) 2013 Guide for preparation of papers LRRD Newsletter

Citation of this paper

Fixed-time artificial insemination in cows and buffaloes using an intravaginal releasing progesterone insert

S E G Noguera, W G Vale**, H F L Ribeiro***, S T Rolim Filho***, A N Reis*, J S Sousa* and A O A Silva*

Graduate Program in Animal Science, Federal University of Pará (UFPA), Belém, Pará, Brazil
* Central de Biotecnologia de Reprodução Animal CEBRAN, Universidade Federal do Pará - UFPA, Rua João Henrique de Carvalho, S/N, CEP 68.741-400 - Castanhal, Pará, BRAZIL
** Instituto de Biodiversidade e Florestas, Universidade Federal do Oeste do Pará – UFOPA, Rua Vera Paz, Campus I – Tapajós, CEP 68035-110, Santarém, Pará, BRAZIL
*** Universidade Federal Rural da Amazônia, Instituto da Saúde e Produção Animal, Setor de Reprodução Animal, Av. Presidente Tancredo Neves nº 2501, Montese, CEP 66.077-530 - Belém, Pará, BRAZIL


In order to demonstrate the feasibility of using fixed time artificial insemination (TAI) in both, bovine and buffalo females by applying an estrous synchronization protocol (i.e., Pregna Heat-E®), results of several works in six comercial farms from different Venezuelan regions were reviewed. Data comprised 219 cows (cows and heifers) and 169 female buffaloes (cows and heifers).


The average observed conception rate of bovine heifers, cows, buffalo heifers and buffalo cows were 41.1%, 39.26%, 0%, and 32.73%, respectively. Conception rates were higher in bovine females than in buffaloes. The overall conception rate for both bovine and buffalo females was 36.5%. In general, strong influences of enviromental-genotype interactions were observed in bovine females. In buffalo females environmental influences also played a clear role on conception rate. In addition, factors associated to age and development in buffalo heifers were also evidenced.  The cost of the protocol Pregna Heat–E™ was slightly higher than others.

Key words: bovine, buffalo, conception rates, oestrus cycle, “Pregna Heat-E”


Using artificial insemination (AI) has been routinely associated to genetic progress and productivity in cattle (Martinez et al 2004). Nevertheless, cattle livestock operations from tropical zones remain poorly developed in comparison to temperate zones (Verde 2012) and the use of AI continues being limited. After approximately 70 years of roughly organized livestock history, the use of AI, genetic progress and productivity in Venezuela is not comparable with countries from temperate zones and well behind from other tropical countries. With the belief of being an easier and cheaper livestock activity, the buffalo cattle livestock has been heavily promoted in Venezuela, during the last 10 years (Scannone 2011).  

Despite the Venezuelan history has been traditionally linked to cattle livestock, an organized functioning and adoption of reproductive technologies such as AI is relatively recent. Today, between 3 to 5 % of Venezuelan cattle operations effectively use AI (personal comunication Dr. Antonio Landaeta-Hernández). Meanwhile in other tropical countries, not much variation is seen. As a continental leader in cattle livestock, Brazil only submitted to AI about 7% of the national dairy herd with a registered frozen semen consumption of 2.845.167 straws (Madalena 2008). After 5 years, the number of dairy cows subjected to AI programs increased 1% only (ASBIA 2011). On the contrary, by the 80´s, 100% of Denmark and France dairy herds were subjected to AI programs while it was 60% in USA (Betteridge 1986).

The greatest milk production input to the Venezuelan market is made by the dual-purpose management system. In this system, productive indicators of the main producer area such as the Maracaibo Lake Basin continues being poor. Here, the average milk yield  ranges from 4.5 to 5.0 kg/day with a total lactation of 1500 to 1700 kg/cow (Chacón and Marchena 2008). Moreover, in this area, reproductive efficiency varies between 50 y 55% (Colmenares et al 2007). With reference to other tropical latinamerican countries such as Brazil and Colombia, the average milk yield/cow/year is reported to be 1138 and 1046 kg/cow/year, respectively (FAO 2004). In contrast, the average milk yield/cow/year in United States was reported to be 8647 (FAO 2004).    

With regard to buffalo species, probably due to some realistic and unrealistic conditions, the Venezuelan buffalo cattle livestock has experienced a considerable growth during the last 10 years (Hoogesteijn et al 2008). Twenty years ago, about 1000 buffaloes were brought from Trinidad for grazing in the Venezuelan Llanos and Delta Amacuro (Coiran 2008). According Perera et al. (2005) the Venezuelan buffalo population in 2005 was about 200, 000. Meanwhile in late 2008, there were more than 350,000 buffaloes, and were present in almost every state in the country with an estimated milk  production of 420,000 liters (6.01% of national milk production) (Coirán 2008). However, as with other bovine  species, Venezuelan buffalo cattle livestock operations do not show clear development features.   

Genetics and concomitantly productive progress are tightly related to the use of AI. However, a successful AI program relies upon several factors which can be summarized as follows: nutritional, environment, and health and welfare associated factors (Coubrough 1985; Suriyasathaporn 1998; Dobson and Smith 2000; Freitas et al 2008; Lopes Gatius 2012). With reference to buffalo livestock, the main difficulties of AI use are represented by achieving an efficient estrous detection (Vale 2007). Nevertheless, the use of protocols for AI that do not require identification of oestrus and time of insemination have contributed to the increased use of AI in bovine and buffalo herds. These protocols aim to synchronize the AI ​​and use it in all animals of the farm, even with those that are not expressing estrus nor cyclicity. Hence, using such protocols may contribute to increase the genetic improvement, and increase of productivity of meat and milk at a faster and more efficient rate (Baruselli et al 2005). 

Thus, the objective of the present study was to demonstrate the feasibility of using fixed time artificial insemination in both bovine and buffalo females by applying an estrous synchronization protocol (i.e., Pregna Heat-E®) in six commercial farms located in different regions of Venezuelan.

Material and methods


The study was conducted between October 2010 and October 2011 in six farms located in three different states of the Bolivarian Republic of Venezuela (i.e., Táchira, Portuguesa, Carabobo). Farms located in Táchira state were: Farm 1, latitude 8° 33'401 60 N and longitude 72° 05'15 99 W at 120 m above sea level within a region described as a humid tropical forest (Temperature= 24-28 °C, rainfall= 1200-2100 mm, relative humidity= 80%). From this farm, 84 animals were selected (22 postpartum cows and 20 heifers; 18 buffalo heifers and 24 postpartum buffalo cows). Farm 2 was located at latitude 7° 30'33 16 N and longitude 71° 57'43 68 W at 247 mrs above sea level within a region described as a tropical rain forest (Temperature= 15-30 °C, rainfall= 2500 mm, relative humidity greater than 70%. From this farm 78 postpartum buffalo cows were selected. Farm 3 is at latitude 7°38’34.52’’N 72° 16’ 46.44’’  W at 810 meters above sea level within a region described as a tropical rain forest (Temperature= 14-26 °C, rainfall= 1.100- 1.600 mm annually relative humidity= 55%. From this farm, 85 postpartum crossbred Bos taurus x Bos indicus cows were selected. Farms located in Portuguesa state (Farm 4 and 5) were at latitude 8° 42'16'' N and longitude 69° 33'52  W at 200 ms above sea level within a region described as a tropical dry forest (Temperature= 26 °C, rainfall= 1,450 mm., Relative humidity= 63%). Twenty three postpartum beef cows were selected from farm 4, whereas 20 heifers and 49 postpartum cows dual purpose were selected from farm 5. Farm 6, located in Carabobo state, was at latitude 10° 26'59 76 N and longitude 68° 19'00 35 at 137 m above sea level within a region described as a tropical dry forest (Temperature= 27 °C, rainfall= 1100 mm., relative humidity= 64%. From this farm, 49 postpartum buffalo cows were selected. 


A total of 388 animals, 219 cows and 169 buffalo cows were used in this study. Inclusion-exclusion criteria for cows and buffalo cows were as follows: good health, body condition score (BCS) of at least 3 (On a scale 1-5; Houghton et al 1990), absence of reproductive disorders, at least 45 d postpartum, age between 20-30 months and body weight not less than 320 kg.  

Feeding and management 

In order to verify the feasibility of the time of insemination (TAI) protocols under different agroecological and management systems, the farms also included variations in management strategies as follows: 

Farm 1: Located in Táchira state. Here, the herd was composed of crossbred Bos taurus x Bos indicus dual-purpose cows and crossbred buffalo cows. Both cows and buffaloes were raised and managed under rotational grazing conditions. Only postpartum cows received supplementation with a mix of Gliricidia sepium + chopped Elephant grass (Pennisetum purpureum), as well as a mix of mineral salt + molasses + bypass fat (Bio-Lac®).

Farm 2: Located in Táchira state. Here, the herd was composed of  crossbred dairy breeds of buffaloes. Feeding and management was similar to Farm 1, except that lactating buffalo cows received an ad-libitum mix of minerals + molasses.  

Farm 3: Located in Táchira state. Here, the herd was composed of crossbred (Predominantly Bos taurus breeding) dual-purpose cows only. In this farm, cows were handled under grazing conditions and supplemented with chopped grass + sorghum silage + concentrate. 

Farm 4: Located in Portuguesa state. Here, the herd was composed of crossbred Bos taurus x Bos indicus dual-purpose cows. In this farm, cows were managed under rotational grazing conditions on Brachiaria humidicola. Permanent access to mineral salts was allowed. During the dry season, animals were supplemented with hay. 

Farm 5: Located in Portuguesa state. Here, the herd was composed of crossbred Bos taurus x Bos indicus beef breeds cows. Feeding was similar to Farm 4.  

Farm 6: Located in Carabobo state. Here, the herd was composed of dairy buffalo breeds only. The animals were raised and managed under grazing conditions. Supplementation with spent barley grain from the beer industry was provided to lactating buffalo cows and calves. 

Type of study and procedures 

In order to achieve the objective, an observational field study with minimal manipulation of environmental and management conditions was conducted.  

At each farm, chosen animals were selected according to criteria previously explained, and protocols for synchronization and timed artificial insemination (TAI) were administered. The commercial protocol named Pregna Heat-E™ with variations according to species (i.e., buffalo and bovine females) and category (i.e., heifer or mature female) was chosen for this study. Likewise, three different AI technicians were used, thus, ruling out that the human factor might interfere directly on the results. Pregnancy diagnosis was performed by rectal palpation 50-60 days after AI.  

Statistical analyses 

In order to determine statistical differences in conception rate among cows and buffalo cows, a chi-square procedure and Fischer exact tests were applied using BioEstat 5.5™ and SAS (2010). The level of statistical significance for all analyses was pre-established as P<0.05. 

Protocols and age groups  

Protocol "Pregna Heat-E" for bovine cows  

Day 0: Application of intravaginal sponge (Pregna-Heat-E™, VIATECA, Venezuela) impregnated with 250 mg of MAP - medroxy-progesterone acetate - PRID, followed by an intramuscular (IM) application of 5 ml Pregna-Heat™ solution (1 mg estradiol / ml, progesterone 5 mg / ml) and 10 ml of Olivitasan plus™ IM (multivitamin containing minerals). Day 6: eCG. (Folligon) 2.5 ml IM (500 IU), prostaglandin 75 mg / animal (½ dose) for the presence of corpus luteum in early treatment. Day 8: intravaginal sponge removal. Day 9: 1mg IM application of EB. Day 10: TAI, 54-60 hours after the PRID (Progesterone Release Intravaginal Device) removal. 

Protocol “Pregna Heat-E" in buffalo cows  

Day 0: application of intravaginal sponge (Pregna-Heat E'' VIATECA, Venezuela) impregnated  with 250 mg of MAP (medroxy-progesterone acetate) followed by an IM application of 5 mL Pregna-Heat™ solution (1 mg estradiol / mL, progesterone 5 mg / ml). Likewise 75 mg of prostaglandin (1/2 dose). 10 ml of multivitamin with minerals (Olivitasan Plus™). Day 9: Removal of device and application of 150 mg (one dose of prostaglandin Veteglan, Ven) 500 IU of eCG (Folligon, Intervet, VEN). Within 48 hours. Day 11: application of 2 ml GnRH Conceptal IM, Intervet, VEN (0.0084 mg buserelin acetate). All animals were inseminated 14-16 hours after the application of conceptal (day 12). 

Protocol "Pregna Heat-E" for bovine  heifers 

Day 0: application of intravaginal sponge impregnated with 250 mg of medroxy progesterone acetate (MAP) + 2.0 mg IM BE + 75 mg of PGF2α (½ dose) + 10 ml of IM Olivitsan plus (multivitamin with minerals). Day 6: IM application of 400 IU of eCG. (Folligon). Day 8: intravaginal sponge removal + Application of 75 mg of PGF2α IM. (½ dose) Day 9: Application of EB 1 ml IM (1mg). Day 10: TAI 52-54 hours after intravaginal sponge removal.   

Protocol "Pregna Heat-E" in buffalo heifers  

Day 0: application of intravaginal sponge impregnated with 250 mg of medroxy progesterone acetate (MAP) + 2.0 mg IM BE + 75 mg of PGF2α (½ dose) + 10 ml of IM Olivitsan plus (multivitamin with minerals). Day 6: IM application of 400 IU of eCG (Folligon). Day 8: intravaginal sponge removal + Application of 75 mg of PGF2α IM (½ dose). Day 9: 2 ml IM application of GnRH Conceptal, Intervet, VEN (0.0084 mg buserelin acetate). Day 10: TAI 52-54 h after intravaginal sponge removal.   

Results and discussion

Conception rates

As shown in Figure 1, there was a marked difference in the conception rate of crossbred (Bos taurus x Bos indicus) dairy heifers among farms 1 and 5.  A higher conception rate (11/20; 55.0%) was found in Farm 5 compared with Farm 1 (6/22; 27.0%). Concerning the low conception rate observed in Farm 1, it could be due to the occurrence of a strong genotype-environmental interaction since Farm 1 is located in a hot-humid region and the herd has a clear predominance of European Bos taurus influence. In contrast, Farm 1 is located in a region with lower relative humidity and the herd is a predominantly Zebu influence. Previous studies point out that heat stress exerts a strong influence on fertility, and especially under tropical conditions, and varies widely among Bos taurus and Bos indicus genotypes (Vale et al 1984; Turner 1980). Moreover, the results obtained in this study are slightly better than those reported by Soto (2008), who used Pregna-Heat-E™ in crossbred zebu and obtained a conception rate of 52.5%.  

As shown in Figure 2, conception rate of crossbred (Bos taurus x Bos indicus) dairy cows was also higher in Farm 1 than remaining farms. Perhaps, the influence of genotype-environmental interactions is the likely reason for these findings. Marked differences in breeding composition and environmental conditions and management were in place when farm conditions were analyzed (Vale et al 1984; Vale 1094; Turner 1980). Using the same protocol in crossbred cows, Gonzáles (2005) obtained quite comparable results.

Figure 1. Conception rates (CR) in dairy crossbred heifers (Bos taurus x Bos indicus) (Farm 1) South Maracaibo lake, Tachira state / Venezuela, and crossbred beef heifers (Bos indicus x Bos taurus), (Farm 5), in the state of Portuguesa / Venezuela, submitted to the protocol "Pregnant Heat-E", (χ²= 3.3429; P= 0.0675).

Figure 2. Conception rate (CR) of crossbred dairy cows (Bos taurus x Bos indicus) submitted to the protocol ("Heat pregna-E") in accordance with the management of farms, located in the states Tachira (Farm 1 and 3) and Portuguesa (Farm 4), respectively, Venezuela. (χ²= 0.7454; P=0.3879)

Artificial insemination in buffalo heifers

With regard conception rate of crossbred dairy buffalo heifers, these were submitted to the Pregna Heat-E protocol during the period corresponding to the adverse season of that region (February to March). Here conception rate was zero (0/18). Reasons for bad results could be related to anatomical aspects of buffalo heifers such as the reduced size of the cervix. This anatomical odd limit the passage of the pipette when performing AI (Vale  1994; Ohashi, 2001).  

Figure 3. Conception rate (CR) in crossbred dairy buffalo cows were submitted to the protocol Pregna-Heat E™, according to the farm
management, from September November 2010 farm 2 (state of Tachira / VEN), and Farm 6 (state of Carabobo / VEN), (favorable
season) and February 2011, a farm 1, south of Lake, state of Tachira / VEN), (unfavorable time). (χ ² = 1.5898, P = 0.2074).

The conception rate of crossbred dairy buffalo cows is shown in Figure 3. Differences among farms are easily explained by the effect of environmental influences because TAI was not performed at similar (e.g. favorable or unfavorable) season in each farm. The phenomenon of reproductive seasonality in buffalo has been shown in various world regions (Mason 1974; Bhatacharya 1974; Roy 1974; Zicarelli 1990; Zicarelli and Vale 2002). Specifically with Farm 1, where a conception rate of 20.8% was obtained in the unfavorable season, results are better than the 15.6% reported by Paiva and Ramirez (2008) during the unfavorable season using protocols with intravaginal devices - Crestar ®, CIDR ® and Pregna Heat –E. 

Moreover, it is possible to say that the results obtained on the Farm 6 (42.8%) were higher because of the difference of management adopted in this property. In Farm 2, the milking is done mechanically, with calf at foot. Meanwhile, at the Farm 3, milking is also mechanically performed but without calf at foot. The blockage in the release of LH by effect of calf presence was reported in cattle by Gonzáles (2005).

Figure 4. Conception rates (CR) in buffalo versus bovine cows submitted to the protocol "Pregna Heat-E", in the Tachira,
Carabobo, and Portuguesa states, VEN; Farms 1, 2, 3, 4, 5, and 6. (Fisher = 0.9624, P = 0.0212, P <0.05).

Table 1. Economic analysis of the total costs for implementing the protocol "Pregna-Heat E™ in cattle and buffaloes belonging to farms located in different states of the Bolivarian Republic of Venezuela, 2011.






‘’Pregna Heat-E’’




















A.I. Technician















BF=Strong Bolivares ; RS =Brazilian Reais; USD=USA dollares; €=Euros

As shown in Table 1, the total cost for applying the Pregna-Heat E™ protocol was slightly higher than was reported in a previous study (Couto 2007) using PRID (i.e., R $ 50.01 and R $ 55.28). Likely reasons for such differences are due to inflation rate and comparative cost of semen in Venezuela and Brazil. When compared with protocols known as “Low cost protocols” (e.g., CL-Synch and Ovsynch), the Pregna-Heat E™ protocol was found to be more expensive also (R $ 77.76  vs R $ 19.0 and R $ 38.67; respectively).



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Received 31 January 2013; Accepted 13 September 2013; Published 1 October 2013

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