Livestock Research for Rural Development 20 (11) 2008 Guide for preparation of papers LRRD News

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A comparative study on evaluation of three synchronization protocols at field level in both cattle and buffaloes

J Gupta*, A Laxmi***, O Vir Singh** and Ashutosh***

* Extension Division
** Veterinary Office
*** Dairy Cattle Physiology Division
National Dairy Research Institute, Karnal, Haryana, India


This study evaluated pregnancy rate and estrus responses to treatment of cows and buffaloes with three different synchronization protocols. Since, the study was carried out at  field level, animals belonging to both the species were selected at random, irrespective of parity, milk yield and body weight. Three different estrous synchronization protocols were evaluated. Treatment included an injection of prostaglandin (PGF2α, 25mg i m) on palpation of CL; status of the estrous cycle was evaluated by rectal palpation and  fixed time insemination (FTAI) was done 72 h post PGF2α injection. Similar was the second protocol in which, in addition to PGF2α administration Gnadotropin (GnRH; 100g) was administered 60 h post PGF2α injection and FTAI was done 72 h post PGF2α. For the third protocol, GnRH was administered at the time of A.I. post PGF2α injection. Three different groups of animals were maintained for three different protocols respectively.


On comparison it was observed that there was no significant difference (P>0.05) in the percentage of pregnant buffaloes when compared between treatments for three different respective protocols. The same was observed with no. of pregnant cows. Similarly, the efficiency of all the three protocols was evaluated for pregnancy rate to first service; it was observed that, the percentage of pregnant buffaloes resulting on application of 1st and 2nd protocol respectively was significantly higher (P<0.05) than the 3rd protocol and the percentage of pregnant cows resulting on application of third protocol was significantly higher (P<0.01) when compared with first and second protocol. There was no significant difference in the percentage of pregnant animals, for both the species irrespective of no. of services when compared between the protocols. The objective of this study was to evaluate results of synchronization to pregnancy rates achieved for cows and buffaloes with PGF2α alone or with GnRH at field level. For the present study five villages were selected. Animals were either primiparous or multiparous.

Key words: buffaloes, cattle, fixed time A.I., synchronization protocol


The gonadotropin releasing hormone (GnRH) and prostaglandin (PGF2α.) method of estrous synchronization has proven to be very successful in synchronizing estrus in cattle and buffaloes (Odde 1990; Lamb et al 2000, 2004; Amaya-Montoya et al 2007). Although many studies have been carried out with PGF2α alone (Stevenson et al 1983; Brito et al 2002; Battista et al 1984) or in combination with GnRH, the use of artificial insemination technique has been successfully used in breeding farms for genetic improvement of animals. In buffalo there is difficulty in identification of estrus manifestations and for application of A.I. at the accurate time. The use of protocols that do not require the identification of estrus, allow for the increase use of A.I. at fixed time and are more efficient in cattle and buffalo for increasing productivity. Studies by Pursley et al (1995) verified that, administration of GnRH after PGF2α injection increases the rate of synchronized ovulation in bovines. It has been observed that when PGF2α is administered on palpation of functional CL., about 60-70% of treated animals, were detected in estrus within 4 days post PGF2α injection (Twagiramungu et al 1995).The pregnancy rate achieved in relation to the usage of different protocols with fixed time artificial insemination in cows and buffaloes was observed to range between 30-50% (Pursley et al 1997; Baruselli, 1997; Baruselli et al 2003). Ovulation and estrus activity after calving are delayed when the positive feed back effects of estradiol on release of LH from the pituitary are reduced due to various factors. Treatment includes hormonal and managemental strategies (Rhodes et al 2003). Treatment of cows that have not been detected in estrus, but have a detectable CL has mainly focused on the use of PGF2α. When CL was detected following palpation of ovaries, estrous response in majority of cows was displayed within 4-5 day post treatment (Plunkett et al 1984; Vasconcelos et al 1999).


Estrus synchronization programs improve reproduction efficiency by reducing the length of breeding and calving seasons and increasing calf weaning weights. Artificial insemination technique can also be used more efficiently. Prostaglandin (PGF2α) causes CL to regress during the responsive phase and a consequent decrease in the levels of progesterone leading to the development of follicles of the next wave (Galina and Orihuela 2007).  Studies carried out by Dhaliwal et al (1988) and Kamonpatana et al (1979) revealed that PGF2α is effective in inducing estrus. In some of the synchronization protocols GnRH has been included along with PGF2α, either single GnRH injection prior to PGF2α (i.e. seven days earlier to PGF2α injection) and for the other one double GnRH injections i.e. seven days prior to PGF2α and second injection , 48h post PGF2α. administration. The later protocol is better known as Ovsynch protocol. Under these two protocols fixed time insemination is done 72h post PGF2α injection (Pursley et al 1997; Jordan et al 2002; Berber et al 2002).


Material and methods 

Indian cattle belonging to different breeds namely Tharparkar, Sahiwal, Crossbred and Murrah buffalo were considered for treatment. The body weights of the cattle ranged between 300-400 kg BW and that of the buffalo ranged between 400-600kg BW. Body weights of the animals below 300 kg were not considered for the study. Animals suffering from clinical reproductive problems were also excluded. A total of 126 no. of animals were considered for application of three different protocols. For the first protocol (Figure 1), 26 buffaloes and 20 cows were selected. They were subjected to rectal palpation. On palpation of CL, 25 mg of PGF2α was administered and were inseminated at fixed time, i.e. 72h post PGF2α administration and the animals were subsequently inseminated 12h post first AI. 

Figure 1.  Prostaglandin and FTAI based protocol  PGF2
α-Prostaglandin   A.I. –Artificial Insemination 1st AI 72h post PGF2α   2nd AI 12h post 1st AI


For the second protocol (Figure 2) 15 cows and 25 buffaloes were selected. On palpation of functional CL, PGF2α was administered and Burserelin (10 g /animal) was injected 60h post PGF2α administration, and FTAI was done 72h post PGF2α injection. 

Figure 2.
Prostaglandin, GnRH and FTAI based protocol PGF2
α-Prostaglandin   A.I. –Artificial Insemination GnRH administered 60h post PGF2α 1st AI 72h post PGF2α ,  2nd AI 12h post 1st AI

For the third protocol 24 cows and 16 buffaloes were selected. The protocol is similar to the second protocol except that, GnRH is administered at the time of A.I.   

Figure 3
 Prostaglandin, GnRH and FTAI based protocol PGF2
α-Prostaglandin   A.I. –Artificial Insemination GnRH administered at the time of 1st AI 1st AI 72h post PGF2α ,  2nd AI 12h post 1st AI

These animals were either repeat breeding or were suffering from reproductive problems like, silent heat, persistent C. L. and also anoestrus condition (as assumed by farmer). By rectal palpation, the status of C.L. and uterine tone could be judged. PGF2α was administered on palpation of functional C. L. and 72h post PGF2α, lysis of C. L. was confirmed. About 90% of the animals were confirmed in heat as observed by swollen vulva and uterine tone on rectal palpation. Cows were diagnosed for pregnancy via rectal palpation on day 60 post A.I. by a trained technician. Pregnancies resulting from A.I. were validated by rectal palpation by a second technician and by calving dates. Statistical analysis of results for number of pregnant animals in response to the three respective protocols applied were by chi-square test and further the results were also analyzed for first service.




It was observed that for all the three protocols, the percentage of pregnant animals ranged between 60-75%. For the first protocol the percentage of pregnant buffaloes and cows was 64.3% and 55% followed by 64% and 73% for the second protocol and 63% and 66% for the third protocol respectively. When results were analyzed by chi-square test there was no significant difference between the results for either protocol/species (Table 1)

Table 1.  Pregnancy rates in buffalo and cow on application of three different  protocols using GnRH and PGF2α/PGF2 α. and FTAI. at 72h post PGF2 α


Buffaloes pregnant

Cows pregnant





 1st protocol





 2nd protocol





 3rd protocol





There was no significant difference between the three protocols when analyzed for total number of pregnant animals for each protocol

The results for percentage of pregnant animals to first service resulting on application of first protocol for buffaloes and cows was 53% (9/17) and 40% (5/11) respectively; followed by 56% (9/16) and 36% (4/11) for the second protocol and similarly for the third protocol it was 50% (5/10) and 62.5% (10/16) respectively. On statistical analysis, it was observed that on application of first and second protocol, no. of pregnant buffaloes was significantly higher (P<0.05) in comparison to the third protocol. In cows, it was observed that on application of third protocol, it resulted in significantly higher (P<0.01) no. of pregnant animals when compared with the first two protocols (Table 2).

Table 2.  Pregnancy rates in buffalo and cow to first service on application of three different protocols using GnRH and PGF2α/PGF2 α. and 72h post PGF2 α


Buffaloes  pregnant
to 1st service

Cows  pregnant
to 1st service

1st protocol





2nd protocol





3rd protocol





*The number of buffaloes confirmed to be pregnant by 1st and 2nd protocol to first service was significantly more (P<0.05) than the third protocol.
**The number of cows confirmed to be pregnant to first service by third protocol was significantly more (P<0.01) than the first two protocols respectively.

Out of a total of 126 no. of animals, 78 number of animals have calved; the calving rate was 61%.


A six year old buffalo which was a repeat breeder for one year conceived and delivered calf on application of first protocol (1st photograph). Similarly a nine year old KF cross breed cow was a repeat breeder. This cow was treated according to the second protocol. A female calf was born. (2nd  photograph).   The dam is producing 16 kg/day.of milk. A ten year old buffalo which did not conceive for six years was treated according to the third protocol. A female calf was delivered. (3rd  photograph). 

1st  Protocol


3rd Protocol

Calves delivered on application of first, second and third protocol respectively

 The body weight of the animals and the calves born were within the normal range.



The major limiting factor for optimum reproductive performance on many farms is failure to detect estrus in a timely and accurate manner. A number of controlled or breeding programs have been developed for synchronizing groups of lactating cattle. Controlled breeding can be directed to cows that pass a corpus luteum test as determined by rectal palpation of the ovaries and for further administering PGF2α to these animals. An important requirement in this study was the presence of functional CL as directed by an expert veterinary officer. It was observed that all the cows and buffaloes were in heat 72h post PGF2α injection. It has been observed that cows in early and late stages of the cycle tend to exhibit heat within 48-72h after PGF2α administration. It has been observed that cows in early and late stages of the cycle tend to exhibit heat within 48-72h after PGF2α administration  ( 01-35 ) .


For all the three synchronization program/protocols, rectal palpation was done to assess ovarian status (presence of CL). The GnRH injection stimulates the release of luteinizing hormone which causes the follicle to ovulate and synchronize ovulation. For testing protocols and adopting a breeding program one must think of drug cost and anticipated success rate. All the estrus synchronization protocols were observed to be effective in achieving desired pregnancy rate in both cattle and buffaloes. In cows the significant difference (P<0.01) observed on application of the third protocol, when compared with the first two protocols with respect to first service, can not be explained. In this study there were various parameters which could not be uniformly assigned to the animals before their distribution to three different protocols. The overall objective was to compare the efficiency of three different protocols. For all the three protocols, the percentage of pregnant animals (60-70%) achieved was observed to be satisfactory for the mentioned species. This study demonstrates accuracy of palpation of functional CL resulting 90% of the animals in heat 72h post PGF2α injection. Benefits of any synchronization program will be obtained only through a systematic, timely and efficient management of events related to reproduction. The results obtained with synchronization program will be significant where poor heat detection system is a limiting factor. The main reason for synchronizing estrus is to facilitate use of AI (Xu and Burton 1999). Till date in a place like India effectiveness of current estrus synchronization strategies is limited, because they rely on visual estrus detection, which is inefficient under field conditions, hence accurate timing for AI is not possible, which leads to decrease in conception rate. The first protocol is for estrus synchronization but reports are available which show that window gap for ovulation time is more. For the second and third protocol GnRH has been included in addition to PGF2α to synchronize estrus and late ovulations occurring beyond 48h. For the third protocol, GnRH may help in survival of conceptus and also by preventing early lysis of CL which is required for maintenance of embryo. The proportion of dairy animals detected with a functional CL at the time of PGF2α administration was 90% and regression of CL 72 h post PGF2α injection was 100%. The 10% of the animals possessed luteal cyst in the ovaries. These animals were confirmed in heat 84h post PGF2α injection in which AI was done 12h post heat confirmation which coincided with the second insemination included for the service. (am-pm rule) conducted for the remaining synchronized animals.


Since, the veterinary officer was an expert and availability an able stock man the time period for hormone administration and fixed time insemination and drug administration was strictly followed, a  pregnancy rate ranging between 60-75% for both the species was obtained . However acceptable results for pregnancy rates to first service in buffaloes were obtained (50-56%). Somehow, in cows there was significant variation when the result for pregnancy rate to first service was compared between the three protocols (36-63%).


Out of total 126 no. of animals, 78 animals have calved and results of calving for the rest of animals is awaited. All the three protocols are prostaglandin based. Fixed time insemination, estrus synchronization protocols are found to be effective, at field level in both the species, with acceptable pregnancy rate. The choice of protocol depends on cost, labor facilities. Success requires a cow herd with a close calving interval, good nutrition, and facilities, high quality semen and accurate heat detection (if heat detection is used).


Cows are bred within 72-84h after PGF2α injection. With improved techniques, different protocols with combination of gonadotropin releasing hormone and prostaglandin have been employed (Pursley et al 1997; Lucy et al 1986; Stevenson et al 1996).


There are different protocols like Ovsynch in which GnRH is injected 7 days prior to PGF2α treatment, causes development of follicles or ovulation of the dominant follicle depending on the stage of the estrous cycle. The second GnRH is administered 48h post PGF2α injection (Bodsteiner et al 1996; Wiltbank 1997; Paul and Prakash 2005; Baruselli et al 1999).  The Co synch protocol is the same as the Ovsynch protocol except that second GnRH is being injected at the time of fixed time A.I. post PGF2α injection (Pursley et al 1995; Geary et al 1998). In GnRH + PGF2α program, GnRH is injected in all the cows and PGF2α is injected at an interval of 7 days post GnRH injection. Animals which are observed in heat 3-5 days post PGF2α injection are inseminated or alternatively they can be inseminated at fixed time i. e. 3days post PGF2α injection. In a modified targeted breeding GnRH + PGF2α program eligible cows receive PGF2α injection and further GnRH is injected 14 days post PGF2α injection and once more they are again injected with PGF2α 7 days post GnRH injection. They all are inseminated 3-4 days post PGF2α injection.


The protocol which is described in this article is an in- house developed protocol, in which only one GnRH injection is administered 60h post PGF2α. It is found to be cost effective where more than one injection of GnRH is required and is less laborious, but at the same time needs trained person for detection of functional CL, it has been reported that administration of PGF2α after ascertaining the status of CL is more economic for achieving acceptable pregnancy rates. Estrus synchronization is a useful technique in cattle practice as it allows the use of fixed time AI or improved heat detection efficiency. Estrus synchronization with fixed time AI eliminates the practical problems of heat detection in dairy cattle.



Amaya-Montoya C, Matsui M, Kawashima C, Hayashi KG, Matsuda G, Kaneko E, Kida K, Miyamoto A and Miyake Y 2007 Induction of ovulation with GnRH and PGF2α at two different stages during the early postpartum period in dairy cows ovarian response and changes in hormone concentrations. Journal of Reproduction and Development 53:867-


Baruselli P S, Madureira E H, Visintin J A, Barnabe V H, Barbane R C and  Amaral R 1999 Timed insemination using synchronization of ovulation in buffalo.

Baruselli P S, Berber R A, Madureira E H and Carvalho N T  2003 Half  dose of Prostaglandin F2 is effective to induce luteolysis in the synchronization of ovulation protocol for fixed time artificial insemination in buffalo (Bubalus bubalis) Brazilian Journal of Veterinary Research and Animal Science. 40 : 397-402 

Battista P J, Rexroad C F and Williams W F 1984 Effects of progesterone administered to dairy heifers on sensitivity of corpora Lutea to PGF2α and on plasma LH concentration Theriogenology 22: 47


Berber RC de A, Madureia E H and Baruselli P S 2002 Comparison of two ovsynch protocols (GnRH versus LH) for fixed timed insemination in buffaloes (bubalus bubalis) Theriogenology 57(5): 1421-1430.


Bodsteiner KJ, Kot K, Wiltbank M C and Ginther O J 1996 Synchronization of emergence of follicular wave in cattle Theriogenology 45: 1115-1128


Brito L F C, Satrapa R , Marson E P and Kastelic J P 2002 Efficacy of PGF2α to synchronize estrus in water buffalo cows (bubalus bubails) is dependent upon plasma progesterone concentration, corpus luteum size and ovarian follicular status before treatment. Animal Reproduction Science 73:23-35


Dhaliwal G S, Sharma R D and Singh G 1988 Efficacy of prostaglandin F2-alpha administration for inducing estrus in buffalo. Theriogenology 29: 1401-1406

Galina C S and Orihuela A 2007 The detection of estrus in cattle rais ed under tropical conditions : What we know  and what we need to know  Hormones and Behaviour 52: 32-38

Geary T, Whitter J C, Downing E R, Lefever D G, Silox R W, Holland M D, Nett F M and  Niswender G D 1998 Pregnancy rates of post- partum beef cows that were synchronized using Syncro-mate-B for the Ovsynch protocol. Journal of Animal Science 76: 1523-1527


Jordan E R, Schouten M J, Quast J W, Belschner A P and Tomarszewski M A 2002 Comparison of two timed artificial insemination (TAI) protocols for management of first insemination postpartum. Journal of Dairy Science 85: 1002-1008


Kamonpatana M, Pansin C, Jetana T, Sophon S, Sravasi S and Srisakwattana K 1979 Factors causing low conception rates when PGF2α is used for oestrous synchronization in Swamp buffaloes. Buffalo Journal Supplementary 1:127-143


Lamb G C, Cartmil J A I and Stevenson J S 2004 Effectiveness of select synch Gonadotropin-releasing hormone and ProstaglandinF2α for synchronizing estrus in replacement beef heifers. Professional Animal Scientist 20:27


Lamb G C, Nix D W, Stevenson J S and Corah L R 2000 Prolonging the MGA- prostaglandin F2α interval from 17 to19 d in an estrus-synchronization system for heifers. Theriogenology 53:691.


Lucy M C, Stevenson J S and Call E P 1986 Controlling first service and calving interval by prostaglandin F2α, gonadotropin releasing hormone, and timed insemination. Journal of Dairy Science 69:2186−2194


Odde K G 1990 A review of synchronization of estrus in postpartum cattle. Journal of Animal Science 68:817-830


Paul V and Prakash B S 2005 Efficacy of the Ovsynch protocol for synchronization of ovulation and fixed time artificial insemination in Murrah buffaloes (Bubalus bubalis). Theriogenology 64: 1049-1060


Plunkett S S, Stevenson J S and Call E P 1984 Prostaglandin F2α for lactating dairy cows with a palpable corpus luteum but unobserved estrus. Journal of Dairy Science 67:380–387


Pursley J R, Mee M O and Wiltbank M C 1995 Synchronization of ovulation in dairy cows using PGF2a and GnRH. Theriogenology 44: 915-923


Pursley J R, Wiltbank M C, Ottobore J S, Garverick H A and Anderson L L 1997 Pregnancy rates per artificial insemination for cows and heifers inseminated as a synchronized ovulation or synchronized estrus. Journal of Dairy Science 80:295


Randel R D, Lammoglia M A and Lewis A W 1996 Exogenous PGF2α enhanced GnRH–induced LH release in postpartum cows Theriogenology 45:643–654


Rhodes F M , McDougall S, Burke C R, Verkerk G A. and Macmillan K L 2003 Invited review Treatment of cows with an extended postpartum anestrous interval. Journal of Dairy Science 6:1876-1894


Stevenson J S, Kobatashi Y, Shipka M P, and Raucholz K C 1996 Altering conception of dairy cattle by gonadotropin-releasing hormone preceding luteolysis induced by prostaglandin F2α. .Journal of Dairy Science 79:402−410


Stevenson J S, Lucy M C, and Call E P 1983 Failure of timed inseminations and associated luteal function in dairy cattle after two injections or PGF2α .Theriogenology 28:937


Twagiramungu H, Guilbault L A, Proulx J G and Dufour J J 1995 Influence of Corpus luteum and induced ovulation on ovarian follicular dynamics in postpartum cyclic cows treated with Buserelin and Cloprostenol.  Journal of Animal Science 72: 1786-1805


Wiltbank M C 1997 How information on hormonal regulation of the ovary has improved understanding of timed breeding programmes. Proceedings of Annual Meeting of the Society for Theriogenology pp.83-97


Xu  Z Z and Burton L J 1999 Reproductive performance of dairy heifers and estrus  synchronization and fixed-time artificial insemination.  Journal of Dairy Science 82:910-917

Received 3 July 2008; Accepted 24 August 2008; Published 6 November 2008

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