Introduction

In dairy cows and buffaloes pregnancy rates are getting reduced due to poor heat expression and detection, anestrus, low conception rates and increased embryo mortality. Furthermore, these impediments to optimal reproductive performance are exacerbated under stressful environmental conditions such as heat stress, which is even more detrimental in higher milk producing cows and buffaloes. Accurate and efficient detection of estrus is a key factor to improve reproductive efficiency in dairy animals. However, cows and especially buffaloes showing seasonal anestrus, silent estrus and subestrus are important constraints in heat detection system. Efficient estrus detection can improve overall reproductive efficiency in dairy animals.  However, proper control of the time of estrus is difficult, since peak estrus activity in most of animals often occurs at night and determination of the actual onset of standing estrus in the herd may be difficult without 24 hrs observation. Therefore, various estrus synchronization protocols have been developed to bring a large percentage of a group of females into estrus at a pre-determined time. Effective use of synchronization protocol in anestrus postpartum animals will increase the proportion of females that become pregnant resulting in shorter calving to conception interval in cattle and buffaloes. Synchronization of estrus implies the induction of estrus to bring the percentage of a group of females into estrus at a short, pre-determined time.  Progestogens and analogs of GnRH and prostaglandins are commonly used to improve fertility in farm animals. Progestogen releasing devices act as artificial corpus luteam (CL) and causes negative feedback on the LH release. Withdrawing the implant 7-9 days after insertion induces LH surge. A number of protocols have been evolved for synchronization of estrus and ovulation. Use of GnRH analogs is based on the assumption that LH responsive follicle would be present in the ovary or potentiate the progesterone release from the existing functional CL. The response of the follicle to GnRH analogs would be ovulation, lutenization of the follicle or no response. Prostaglandin analogs are the major luteolytic agents but it will not cause luteolysis within 5 days after CL formation and used only in cycling animals or those treated with GnRH at least 7 days earlier.  Estrus synchronization brings a group of cows and buffaloes to heat within a period of 36 to 96 hours whereas synchronization of ovulation is aimed at inducing ovulation within 16-20 hr after GnRH administration. The latter technique is mainly coupled to fixed time AI (FTAI) program as it avoids heat detection. A brief description of the progesterone, GnRH and PGF based protocols is listed below.

Synchronization of Estrous Cycle by Shortening of the Luteal Phase

Prostaglandin Based Protocols

Prostaglandins based protocol approach Prostaglandin (PG) is a naturally occurring hormone. During the normal estrous cycle of a non-pregnant animal, PGF is released from the uterus 16 to 18 days after the animal was in heat. This release of PGF functions to destroy the CL. The CL is a structure in the ovary that produces the hormone progesterone and prevents the animal from returning to estrus. The release of PGF from the uterus is the triggering mechanism that results in the animal returning to estrus every 21 days. Commercially available PGF2α (Lutalyse, Estrumate, Prostamate) gives the herd owner the ability to simultaneously remove the CL from all cycling animals at a predetermined time that is convenient for heat detection and breeding Patterson et al. (2003). The major limitation of PGF₂α is that it is not effective on animals that do not possess a CL. This includes animals within 6 to 7 days of a previous heat, pre-pubertal heifers and postpartum anestrous cows. Despite these limitations, prostaglandins are the simplest method to synchronize estrus in cattle. Synchrony of estrus and fertility with these products is good with cyclic females, such as virgin heifers, but cannot induce estrous cycles in non-cycling cows.

One Shot Prostaglandin Injection Protocol

A single injection of prostaglandin is given to cyclic females and then these females are bred as they express estrus. The disadvantage of this program is that one-third of the females do not respond to the injection. After single PG injection, approximately 70% of cows and buffaloes (those on days 6 or greater of the cycle at injection) come into heat within 2 to 5 days of estrous cycle. The animals detected in heat are inseminated 12 hours after heat detection or inseminated at 72 to 96 hrs without reference because majority of the animals come into heat between 48 to 72 hrs after PG injection. This protocol represents the greatest savings in cost and labor associated with treatments because only one injection is given and not all the cows need it.

Two Short Prostaglandin Injections Protocol

The two injection programs for synchronization with PG are intended to increase the proportion of females with a CL that is responsive to regression with PG. Usually, the recommendation was that two injections of PG to be administered 11 days apart, but recent data suggests that a 14-day interval is more effective.

Programme 1

Initially, an injection of PG is given to all cows. Two to five days after the first injection, ~70 % of the cyclic females should be in heat. Females detected in heat are inseminated 12 hours after the heat. The animals not inseminated after the first PG will receive a second PG injection 11 to 14 days later and are inseminated 12 hours after detection of heat (Fig. 1). This programme possibly helps to save the extra PG injection in those animals came into heat following first PG injection.

PG                                                                          PG

Detect estrus AI               If no AI                                    detect estrus AI

0                      5                      Day                            11                    15

Fig. 1: Two injections PG preprogrammed with breeding after both injections. (Program 1) or only after the second injection (Program 2)

Programme 2

In this programme PG was given to all animals but no inseminations are performed. Again second PG was administered 11/14 days after the first PG and the animals are inseminated 12 hours after detection of heat. This regimen theoretically synchronizes estrus in 100% of cyclic cows within two to five days after the second PG. In application, some cows that exhibit heat after the first injection fail to show after the second PG, and anyway synchronization responses of 75 to 85% after the second PG are common with this protocol. It has also been shown that if they did AI twice at a fixed time of 72 and 96 hrs after the second injection CR are comparable with those following AI at spontaneous estrus (Fig. 1.1). In this programme have lowers expense and handling, but results in two synchronized groups instead of one and a longer breeding period Sahatpure et al., 2008.

PG    No detection of heat & AI                                         detect estrus &AI

0                                       Day                                                 11                   15

Fig. 1.1: Two injection PG programme with breeding after the second injection

Synchronization of Estrous Cycle by Extending the Luteal Phase

Progesterone Based Protocols

Synthetic progestogen preparations, available for oral (MGA), subcutaneous (Synchro-Mate B, Crestar ear implants) or intravaginal (PRID, PRID DELTA, CIDR-B, Cue- Mate and TRIU-B) administration have been used effectively to synchronize the estrus in cyclic as well as for inducing estrus in acyclic dairy animals. After insertion of the P₄ implant in both cyclic and acyclic cattle and buffaloes, leads to an increased circulatory concentration of P₄, which exert a negative feedback to higher centres i.e. hypothalamus and anterior pituitary. P₄ enhances the hypothalamic sensitivity to estrogen with subsequent increase in the intensity of heat. Sudden withdrawal of these devices (7-9 days later) leads to an abrupt decline of P₄ concentration in the circulation and promoting the release of GnRH as well as FSH and LH with subsequent resumption of ovarian cyclicity. Moreover, one extra PG injection is essential one day before implant removal in cyclic animals for the intent to complete lysis of natural CL. These events almost mimicking like a natural luteolysis mechanism. Commonly, the buffalo exhibits the estrus signs within 2-5 days after device withdrawal depending on the size of largest follicle at the time of implant removal. The animals at detected heat are inseminated 12 hours after heat detection or FTAI after 48 to 72 hrs after PG injection. Administration of eCG on the day Singh et al (2006) or one day Murugavel et al (2009) before the removal of CIDR implant improve the ovulation rate and pregnancy rate in anestrus cows and buffaloes. Administration of eCG probably helps to complete the recovery of the HPG axis function already stimulated by the P₄ treatment. In general, CIDR, PRID, TRIU-B or Crestar based treatment regimens resulted in 80-100% estrus induction with 30-60 % conception rate (CR) following FTAI depending on the season (breeding & non breeding) and cyclic status when treatment was initiated. However, the CR was considerably low in acyclic animals during summer due heat stress. Diagrammatic representation of progestogen based protocols in cyclic and acyclic animals was given in Fig.1 and 2, respectively.

A                                              PG+ PMSG(O)   A

In                                              500 IU            Out     FTAI     FTAI

0                      DAY                      7                 8            9           10

A= Crestar/ CIDR-B/ TRIU-B; (O)-Optional

Fig. 2: Progesterone implant schedule for estrus synchronization in cyclic cattle & buffalo

A                                                       PMSG      A

In                                                      500 IU      Out   FTAI     FTAI

0                      DAY                               7         8           9          10

A= Crestar/ CIDR-B/ TRIU-B

Fig.2.1: Schematic illustration of P₄ implant schedule in true anoestrus cattle & buffaloes

Manipulation of Follicular Waves And Luteal Lifespan Through Administration of GnRH-PG

GnRH based protocols are primarily developed for estrus and ovulation synchronization in cyclic cattle and buffaloes for better reproductive herd management. Acyclic buffaloes essentially require P₄ priming in order to induce overt, ovulatory and fertile estrus. In this process, GnRH supports final growth and maturation of ovulatory follicle leading to ovulation through release of pituitary gonadotrophins. Administration of GnRH, thus, can cause ovulations or luteinization of the dominant follicles (DFs), which should provide the necessary P₄ priming to anestrus females for experiencing overt estrus following therapy Sharma et al. (2009).

Ovsynch

Ovsynch protocol can be used in cyclic as well as in acyclic animals. Pursley et al (1995) at the University of Wisconsin, Madison, developed a protocol, called ‘Ovsynch’, consists of an initial injection of GnRH at random stage of the estrous cycle, which causes ovulation or luteinization of the large follicle(s) present in the ovary and synchronizes the recruitment of follicles for a new wave. Seven days later, an injection of PG induces regression of the CL, which facilitates final maturation of the synchronized DF. A second GnRH injection, given 16-20 hrs after PGF₂α, induces ovulation of the DF with precise synchrony amongst treated animals to allow timed inseminations after the second GnRH. (Fig.3). Lactating dairy cows subjected to ovsynch have lower CR because ovulation is not adequately synchronized in approximately 35% of the animals Colazo et al. (2009).This variability in ovulation synchronization may be due to the stage of the estrous cycle at the time of first GnRH in ovsynch regimen. Commencement of the Ovsynch protocol during metestrus (days 1 to 4 of the estrous cycle), when the DF is not adequately developed to ovulate in response to first GnRH, will results in reduced pregnancy rate. Similarly, initiation of Ovsynch protocol during late diestrus (day 13 to 17), when the CL is going to regress, will result in premature ovulation before FTAI. Finally, if the Ovsynch regimen starts during proestrus (days 18 to 21), the first injection of GnRH will induce ovulation but the formed CL might not respond to the injection of PG 7 days later. Therefore, Ovsynch treatment was effective when it is initiated between day 5 and 12 of the estrous cycle or the DF size more than 10 mm at the time of first GnRH injection Vasconcelos et al. (1999).

The Ovsynch protocol has been used primarily in cyclic buffaloes with CRs ranging from 33 to 64% after TAI Berber et al. (2002); Paul and Prakash (2005). However, CR following Ovsynch in acyclic cattle (9 to 37%) and buffaloes (0 to 50 %) is highly variable. The ovulatory response to GnRH injection is a major limitation in Ovsynch regimens, to achieve an optimal CR in both cyclic and acyclic animals. Follicular diameter at the time of GnRH injection is an important determinant to predict the ovulatory response. Hence, the large follicle diameter (LFD) is >9-10 mm at the time of GnRH is prerequisite for the success of ovsynch programme in anestrus dairy buffaloes Rohilla et al. (2003). At the time of GnRH administration, stage of follicle development (recruitment, growing, dominant or ovulatory) is not known, leading to poor ovulatory and fertility response which is the major limitation of the ovsynch protocol.

GnRH                                                  GnRH

2.5 ml                                 PG            2.5 ml           FTAI

16-20 hr

0            DAY                       7                   9                 10

Fig.3: Schematic representation of treatment schedule of Ovsynch protocol

Presynchronization Protocols

A battery of presynchronization protocols has been developed to achieve an optimal dominant follicle size (>9-10 mm) or to bring all the animals in the window of day 5 and 12 of estrous cycle at the time of first GnRH injection in Ovsynch protocol.

Presynch-Ovsynch

A Presynch protocol is desirable prior to Ovsynch because it presynchronizes estrous cycles, so when the timed A.I. protocol is initiated, the majority of cows are between days 5 and 12 of the estrous cycle, an ideal time to start the Ovsynch protocol to maximize fertility. This protocol involves the administration of two PG injections at 14 days apart and the initiation of Ovsynch 12 days after the second PG treatment. The P/AI was considerably higher in cows treated with “Presynch-Ovsynch” than Ovsynch (49 vs. 37%, Moreira et al. (2001); vs. 37%, El-Zarkouny et al. (2004).

GnRH                                          GnRH

PG                               PG                     2.5 ml                       PG              2.5 ml             FTAI

14 d                             12 d                           7 d                   2 d          16-20 hr

Fig.4: Schematic representation of treatment schedule of Presynch- Ovsynch protocol

G6G/Ovsynch

Scientific rationale in G6G/Ovsynch is similar to presynch-Ovsynch to bring all the animals in the window of day 5 and 12 of estrous cycle at start of Ovsynch protocol. This protocol is a kind of presynch-Ovsynch, consists of an initial PG injection on any stage of estrous cycle (day 0) followed by GnRH injection 2 days later and Ovsynch initiated 6 days after the first GnRH injection. Following treatment with G6G/Ovsynch, the ovulatory response to first GnRH in Ovsynch (84.6%) was higher than the control Ovsynch cows (53.8 %). Moreover, 92 % of G6G/Ovsynch cows were successfully synchronized to timed-AI compared with only 69 % of Ovsynch cows. Moreover, pregnancy per AI tented to be higher in G6G/Ovsynch protocol (50%) than the traditional Ovsynch regimen (27%), Bello et al. (2006). Ribeiro et al. (2011) also observed the similar result by using large sample size.

GnRH                     GnRH                                           GnRH

PG        2.5 ml                      2.5 ml                         PG            2.5 ml           FTAI

2 d                6 d                          7 d                                            16-20 hr

Fig. 5: Schematic representation of treatment schedule of Presynch Ovsynch protocol

Ovsynch-Plus

To ensure the largest follicle diameter >9 mm at the time of first GnRH injection in Ovsynch schedule, Sharma et al (2004) developed a novel protocol named as Ovsynch-Plus by injecting PMSG (500 IU) 3 days prior to Ovsynch (Fig. 6). Administration of PMSG enhances the dominant follicle size > 9mm and it ensure consistently similar ovarian follicular picture of all animals at the time of first GnRH injection. The protocol consists of administering a small dose of PMSG (Folligon, 400 IU) on day 0, followed by GnRH on day 3, luteolytic dose of PGF_2α on day 10, and second injection of GnRH on day 12, followed by FTAI at  6 and 30 h later.  These regimens yielded estrus induction and conception rate of 80 and 30%, respectively in true anestrus buffaloes following FTAI. Following Ovsynch plus treatment in peripubertal buffaloes, 50% conception rate was noticed Sharma et al. (2009).

PMSG      GnRH                                                       GnRH

400 IU       2.5 ml                                   PG       2.5 ml (AI)   AI

0                 3           DAY                           10                  12         13

Fig. 6: Schematic representation Ovsynch-Plus regimen

Double Ovsynch

Double-Ovsynch is a novel pre-synchronization protocol with intent to achieve a more synchronous stage (days 5-12 of the cycle) of the cycle at the initiation of the Ovsynch protocol which consists of GnRH on day 0, followed by an injection of PG 7 day later and GnRH 3 day after PG, then began the Ovsynch-TAI protocol 7 day later. Double-Ovsynch had greater P/AI (49.7%) than Presynch (41.7%). Moreover, Double-Ovsynch increased P/AI only in primiparous (65.2% vs 45.2%) and not multiparous (37.5% vs 39.3%) cows Souza et al. (2008).

GnRH                                 GnRH                GnRH                          GnRH

2.5 ml                 PG           2.5 ml                 2.5 ml               PG      2.5 ml               FTAI

7 d                    3 d                 7 d                  7 d                2 d            16-20 hr

Fig. 7: Schematic representation Double Ovsynch regimen

Modified Ovsynch Protocol

Baruselli et al. (2002) developed a new pre-synchronization protocol by pre-synchronization with GnRH 7 days before the beginning of GnRH/PGF₂α/GnRH treatment will increase the number of buffaloes with dominant follicles that respond positively to the first GnRH of traditional treatment. Following treatment CR was in modified Ovsynch (56.0%; 61/109) than in traditional Ovsynch (39.5%; 45/114). The same regimen was applied in anovular buffaloes and the CR during FTAI was 21.4%.

GnRH                                GnRH                                                   GnRH

2.5 ml                                2.5 ml                              PG                 2.5 ml       FTAI

16-20 hr

-7                                          0            DAY                   7                    9                        10

Fig. 8: Schematic representation modified Ovsynch regimen

Doublesynch

To improve the ovulation rate after first and second GnRH in Ovsynch regimens, Cirit et al. (2007)developed a new synchronization method called as ‘Doublesynch’ that consists of an extra PGF_2α injection 2 days prior to initiation of Ovsynch protocol (Fig. 9). This new program resulted in better ovulation rate following first GnRH injection (88.9%) and increased the pregnancy rates by 22.2 percentage (50.0% vs. 72.2%). Öztürk et al. (2010) applied this protocol in anestrus dairy cows and they reported 72% (18/25) pregnancy rate. Mirmahmoudi and Prakash (2012) evaluated this protocol in buffaloes and they reported pregnancy rates of 60% (12/20) in cyclic and 55% (6/11) in anestrus buffaloes (Souza et al., 2008).

GnRH                                         GnRH

PG     2.5 ml                        PG            2.5 ml                      FTAI

16-20 hr

-2           0         DAY           7                   9                              10

Fig. 9: Schematic illustration of Double synch treatment schedule

Estradoublesynch

In view of the cost effectiveness as well as augmenting the ovulatory response to first GnRH in heatsynch regimen, developed a new synchronization protocol called Estradoublesynch, which consist of an extra PGF_2α injection 48 hr before commencement of the heatsynch protocol. They documented that higher pregnancy rate following Estradoublesynch regimens in cyclic (62%) and acyclic (64%) buffaloes.

Modifications of Ovsynch Protocol

Cosynch

Co-Synch is a precise form of Ovsynch in which FTAI occurs at the time of the second GnRH injection. The main advantage of Co-synch is that one less handling is required for each cow compared to Ovsynch; the disadvantage is that CR slightly lower than the Ovsynch, but pregnancy losses significantly lower resulting in similar calving rates to the Ovsynch program.

GnRH                                                       GnRH&

2.5 ml                                    PG                FTAI

0            DAY                       7                       9

Fig. 10: Schematic illustration of Cosynch treatment schedule

Select Synch

Select Synch is a breeding option for those herds with good heat detection programme and that prefers to breed cows based to standing estrus.  In this programme the animals are inseminated at detected estrus after PG injection. It saves an additional injection of GnRH.

GnRH

2.5 ml                                 PG

Estrus detection &AI

0            DAY                       7

Fig.11: Schematic illustration of Select synch treatment schedule

Hybrid Synch

Hybrid Synch is protocol in which AI was carried out the animals at detected estrus after PG and the remaining animals was carried out FTAI at day 10.

GnRH

2.5 ml                                 PG   Estrus detection      FTAI

&AI

0            DAY                      7                                   10

Fig.12: Schematic illustration of select synch treatment schedule

Heatsynch

Heat-Synch is an substitute to Ovsynch in which 1.0 mg of estradiol cypionate (ECP) or estradiol benzoate (EB) is administered 24 hours after the PGF₂α injection of Ovsynch to induce ovulation rather than administering GnRH 48 hours after PGF₂α. Cows receiving EB or ECP usually exhibit 100% estrus behavior as well as uterine tone. The main advantages of using Heatsynch was lower cost compared with GnRH. Overall, Heat-Synch results in similar reproductive performance to Ovsynch but may not be effective for synchronizing acyclic animals.

GnRH                                             EB/EC

2.5 ml                                 PG       (1mg)              FTAI

0            DAY                       7            8                        10

EB- Estradiol Benzoate; EC-Estradiol cypionate

Fig.13: Schematic illustration of Select synch treatment schedule

Resynchronization Protocols

Protocol 1

For better reproductive management of dairy herd involves early diagnosis of non-pregnant animals after breeding and shortly resynchronize them with Ovsynch or CIDR protocols. This programme is applied to cows and buffaloes that are already inseminated at FTAI (day 0) and found to be non pregnant on day 33 post AI and Ovsynch protocol was initiated to resynchronization of those animals (Fig. 14).

PD (NP)

FTAI                                GnRH                 PG     GnRH   FTAI

0            DAY                       33                      40          42       43

Fig. 14: Resynchronization protocol with commencement of Ovsynch on day 33 post AIPD-Pregnancy diagnosis; NP- Non pregnant

Protocol 2

This protocol is applied to animals that are already inseminated at FTAI (day 0) and all such animals are received GnRH injection on day 26 post AI and pregnancy diagnosis is made on day 33 post insemination and found non-pregnant are given PG injection followed by second GnRH injection on day 35 and FTAI on day 36 which saves 7 days in comparison with the former protocol (Fig. 15).

PD (NP)

FTAI                                  GnRH               PG      GnRH FTAI

0            DAY                       26                     33          35        36

Fig. 15: Resynchronization protocol with initiation of Ovsynch on day 26 pos

Summary and Conclusion

Manipulation of estrous cycle done in cattle and buffalo for synchronization of estrus and ovulation. Analogs of GnRH and PGF as well as controlled progesterone release drugs are the mainstay tools. The main limitation in GnRH based protocol is that the ovulatory response of the largest follicle is difficult to predict at the initiation of treatment as it is difficult ascertain its functional status (growing, static or regressing).Further, the response of heifers to GnRH is poor. Main limitation of long progesterone exposure is due to altered steroidogenesis of the follicle and decreased oocyte competence to achieve optimal pregnancy rates with any synchronization protocol, cows should be in good body condition (BCS≥3) and treatments should be initiated only when cows are at least 50 days postpartum.

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