Fixed Time Artificial Insemination in Buffaloes

Artificial insemination (AI) is a proven tool to promote genetic improvement in farm animals, but because of poor estrus detection [1], this technique has limited application in buffalo herds. Moreover, the small number of tested bulls compared to cattle and the low conception rates obtained in AI programs has discouraged buffalo farmers to adopt this technique. For these reasons, the genetic improvement of the buffalo species is running slow.

The first step in breeding and selection activity with regards to dairy animals is the daily recording of milk production and the productivity of each animal. This step together with the progeny testing performed on young bulls, to select sires for use in AI breeding programs, are considered important means to attain the genetic improvement of a species. A consistent number of milk recorded buffaloes is found in countries with the highest presence of this species (India and Egypt), but the highest proportion of recorded milk producing buffaloes is found in Italy, although the number of buffaloes represents less than 10% of dairy animals [2]. In Italy, where accurate production records are kept, and the land where there is the highest proportion of milk producing buffaloes on record, buffalo farming has reached remarkable productive standards (average milk production recorded in 2015 was 2252 kg per lactation, with 7.93% fat and 4.68% protein content) [3].

Despite the remarkable productive standards obtained, the average yield per buffalo head did not improve to the same extent as that of the dairy cattle breeds for which a systematic genetic improvement is carried out and high value proven semen is utilized. The success of genetic improvement programs is essentially based on the utilization of AI in the field. This necessary condition yielded suboptimal results in buffalo breeding mostly due to the incorrect time of insemination, on account of poor estrus expression [1,4,5]. Estrous behavior in buffaloes has a lower intensity than in cows and is therefore much more difficult to detect [1]. Acceptance of the male by the female in heat is considered as the most reliable estrus indicator [6,7]. Frequent urination, bellowing, vulvar swelling, and mucus discharge, are referred to be salient signs of estrus in cows but their expression in buffaloes is extremely weak [6]. Ovulation cannot be predicted from estrus behavior signs because of a wide variability in the interval between the start of standing estrus and the LH peak [8] and consequently ovulation time, which usually occurs 35.5 h after the LH peak in spontaneous estrus [9]. For these reasons, the use of AI is limited in buffaloes, considering that a high conception rate depends mainly on insemination at the correct time relative to ovulation [5,6]. Moreover, although buffaloes are polyestrous, their reproductive efficiency shows wide variation throughout the year. Buffalo cows exhibit a distinct seasonal change in displaying estrus, conception rate and calving rate, therefore buffaloes calving during an unfavorable season may not resume ovarian activity until the following favorable season [4]. This seasonal reproductive pattern affects pregnancy rate when AI is utilized outside of the breeding season.

Hormonal treatments may overcome some of the difficulties due to estrus detection and seasonality, increasing the utilization of AI in buffaloes [4]. In this chapter, the different approaches for estrus synchronization and fixed time insemination are explained.

Protocols for Fixed Time AI

It is essential for fixed time inseminations that estrus and ovulation be synchronized by the exogenous administration of hormones. Controlled breeding programs that do not require the identification of estrus have been studied in cattle [10-12]. Estrus synchronization approaches in buffalo [13] have been adopted from those in cattle, either

1. Manipulating peripheral progesterone concentration or
2. Manipulating follicular growth and timing of ovulation

Manipulating Peripheral Progesterone Concentration

The corpus luteum (CL) formed on the ovary following ovulation produces progesterone, which exerts negative feedback on the release of gonadotrophin so that the endocrine events leading to the maturation of the preovulatory follicle and succeeding ovulation are inhibited until progesterone drops following the regression of CL [6]. Thus, by controlling the lifespan of the CL, it is possible to control ovulation and synchronize estrus. Two approaches can be used to control or mimic the lifespan of the CL:

1. By inducing premature luteolysis (prostaglandins);
2. By simulating CL function (long-term administration of progesterone or progestogens, followed by sudden withdrawal).

1. Induction of Luteolysis

Initial estrus synchronization programs focused on altering the estrous cycle by regression of the CL with the administration of prostaglandin. Prostaglandin F2 alpha (PGF2α) is the key molecule, secreted from the uterus, responsible for regression of the CL in several species including buffaloes [14-16], in the event that the animal did not conceive. In buffaloes, it has been reported that circulating progesterone levels decreased within 1 h of PGF2α treatment and evidence of apoptosis was demonstrable at 18 h post treatment [16]. Commercially available PGF2α preparations administered during the mid-luteal phase (Day 5 to 17 of the estrus cycle), in the presence of a functional CL, will result in luteolysis, decrease in progesterone and return to estrus within 2-3 days. The response of buffaloes to administration of PGF2α would depend upon progesterone plasma concentrations and CL size before treatment. It was found that buffalo cows that failed to ovulate after PGF2α treatment had lower plasma progesterone concentrations and a smaller CL area before treatment as compared with cows that ovulated following treatment; moreover, the interval from treatment to ovulation would depend upon follicular status before treatment [17].

Similar to protocols utilized in cattle, prostaglandins have been administered to buffaloes as a single injection (one-shot method) or two injections separated by 11-14 days (two-shot method) [6,12].

PGF2α One-Shot Method

In this approach only buffaloes having a functional CL, i.e.in days 5-17 of the estrous cycle, can be treated with prostaglandins. Studies in cattle showed that cows treated with a single dose of PGF2α when an active CL was present, returned to estrus within 2-3 days [18,19]. In buffaloes, it was found that the response to the treatment with a single injection of PGF2α was similar to that of cattle [20-22]. Moreover, estrus behavior and endocrine change after the PGF2α induced luteolysis appeared similar to those occurring during natural estrus [22,23]. The one-shot method has the disadvantage that animals have to be palpated or scanned with ultrasonography prior to be treated with PGF2α [24]. Due to the poor palpable characteristics of the buffalo CL, the use of PGF2α might pose difficulty when an ultrasound scanner cannot be utilized.

PGF2α Two-Shot Method

Administration of two doses of PGF2α 11 days apart (Fig. 1) offers the possibility to synchronize estrus treating buffaloes at random, without reference to their exact ovarian status [6], similar to the protocol developed for cattle [25,26]. All females scheduled to be synchronized received PGF2α on Day 0 and on Day 11 of the treatment. The basics of this schedule is that at the time of the 1st injection of PGF2α only those animals that have a functional CL (i.e. day 5-17 of the cycle) will be responsive. These animals will ovulate, and at the time of the 2nd PGF2α injection, they will be at about Day 8 of the next cycle. The animals that did not respond at the 1st injection (i.e. those between Day 18 and Day 4 of the cycle) should have a responsive CL at the time of the 2nd injection. Hence, all animals will be in the mid-luteal phase at the time of the 2nd injection of PGF2α [6]. Various authors have utilized the two-shot method in estrus control in buffaloes, often using an 11 day interval between two consecutive doses [21,22,27,28].

Figure 1. Estrus synchronization scheme based on induction of luteolysis: double PGF2α injection. Buffaloes can be inseminated at 72 and 96 h from the 2nd prostaglandin injection.

Using PGF2α treatment, buffaloes are inseminated either at fixed times (72 and 96h after 2nd PGF2α treatment) or at observed estrus [22,28,29]. The major limitation of any of these prostaglandin systems is that it is necessary that a functional CL be present for PGF2α to be effective; therefore, PGF2α is ineffective in inducing estrus in prepubertal buffalo heifers and in anestrus buffalo cows because they will not have a functional CL.

Because buffaloes are seasonal breeders, the main limiting factor for utilizing only a PGF2α protocol to synchronize estrus, is the period of the year during which it is used. Buffalo cows exhibit a distinct seasonal change in displaying estrus, conception rate and calving rate. The proportion of buffaloes showing estrus during the period of short-day length is significantly greater than during the period of long-day length, indicating that decreasing daylight is a stronger determinant of resumption of ovarian activity [4,30,31]. Chohan et al., [32] synchronized buffaloes with PGF2α and reported a fertility rate at AI of 22.8% during the low breeding season and 53.3% during the peak breeding season, concluding that the use of PGF2α to synchronize estrus should be done in animals with a functional corpus luteum and preferably during the peak breeding season. These data were also confirmed in a subsequent study in which different doses of PGF2α were compared: buffaloes were treated with 125 μg and 500 μg cloprostenol, conception rates (CR) of 47.8 vs 53.1% were obtained during the peak breeding and 23.5 vs 25.6% during the low breeding season; CR were different between seasons but not between the treatments within the same season [33]. Nevertheless, Sahasrabudhe and Pandit [34] reported that a high percentage of subestrus buffaloes expressed estrus after PGF2α treatment during the hot season. In a large trial on 300 Egyptian buffaloes, the administration of two PGF2α injections 11 days apart resulted in CR (by AI at detected estrus) of 53%-70% with different molecules of PGF2α (dinoprost tromethamine, cloprostenol sodium and luprostiol) used [28]. Neglia et al., [35] utilizing a synchronization treatment with prostaglandin reported a CR at AI of 43.4% during the period of transition to seasonal anestrus. Recently, Pandey et al., [36] reported that the administration of buserelin acetate or hCG on Day 12 post-ovulation has a beneficial impact on CR at AI, after estrus synchronization with PGF2α (52.9 vs 28.6% respectively with the addition of buserelin acetate or hCG and with PGF2α alone). The utilization of an analogue of PGF2α is also reported to synchronize estrus in cycling buffalo heifers [27,37], but the CR at AI was variable ranging from 12.5% to 62.5%.

The detection of estrus after prostaglandin treatment had posed problems because external signs of estrus were found by some workers to be less apparent than at spontaneous estrus. Baruselli, [38] detected a greater variation in the duration of estrus manifestation (36 to 96h) after the administration of prostaglandin. Moreover, he found that the phase during which prostaglandin was administered interfered in the interval from administration and the beginning of estrus signs and ovulation, due to the variation in follicular population at the moment of CL regression. In fact, a large variability is reported in the mean interval from PGF2α treatment to estrus (48-144h) and from PGF2α treatment to ovulation (60-156h) [17]. Consequently, detection of estrus must be optimal in order to identify the appropriate time for AI. Protocols using fixed time insemination and prostaglandin only treatment, cannot be efficient due to the variability in the interval between treatment, estrus and ovulation [13,38,39].

2. Use of Progestins

Estrus synchronization programs involving the use of exogenous progestins aim at preventing estrus from occurring during their administration. Exogenous progesterone or its synthetic derivatives (progestins) inhibits the secretion of gonadotropins from the pituitary gland, it inhibits follicular maturation and ovulation and prevents an animal from coming into estrus until its removal [6]. The use of progestins in buffaloes has been shown to be useful in inducing fertile estrus in non-cyclic heifers, to increase fertility during the low breeding season, to reduce the inter-calving period in the post-partum and to synchronize estrus for fixed time AI [4]. Progestins can be administered orally (melengesterol acetate), or given subcutaneously in a polymer (hydron) ear implant (norgestomet), or as a more practical method in an intravaginal device impregnated with progesterone (PRID/CIDR). In order to randomly synchronize a group of females without regard to the stage of the estrous cycle, it is necessary to treat them with progesterone/progestin for a period corresponding to the length of the natural luteal phase; otherwise, the CL might live longer than the duration of the progesterone/progestin treatment [6]. However, the long-term progestin treatment (i.e. 16 days like a natural luteal phase) leads to a low conception rate, probably due to the adverse effects on the intrauterine environment. Therefore, a short-term treatment (7-12 days) is preferable; however, in this case it is necessary to include a PGF2α treatment in order to eliminate any functional CL at the time of device removal [40].

Melengestrol Acetate (MGA)

Melengestrol Acetate (MGA) is a synthetic analogue of progesterone that is orally active and is administered in the feed [41]. However, oral administration of MGA presents a problem for drug dosage monitoring because it seems difficult to ensure uniform daily consumption of feed. Estrus synchronization could be variable if oral consumption of MGA is not uniform. The possibility of milk contamination with progestin residues limits its use in prepubertal heifers only. The most effective system for synchronization of estrus in heifers involves feeding MGA for 14 days, at a rate of 0.5 mg/head/day, and injecting PGF2α 19 days after the last day of MGA feeding. Longer feeding periods of MGA (14 days) always have been associated with low fertility at the first synchronized estrus, but subsequent conception has been normal. Feeding MGA for 14 days followed by injecting PGF2α on Day 19 prevented reduced conception problems [42]. Research in the last decade led to the development of protocols using MGA associated with PGF2α and GnRH: the MGA Select and 7-11 Synch protocols [43]. The two protocols differ in length of treatment (MGA Select - 33 days; 7-11 Synch - 18 days) as well as the length of interval to estrus and resulting synchrony of estrus; however, there were no differences reported in pregnancy rates between these protocols among cows inseminated on the basis of observed estrus. Peak estrus response among cows assigned to the MGA Select protocol typically occurs 72 hours after PGF2α. Pregnancy rates were optimized for cows assigned to the MGA Select protocol when fixed-time AI was performed at 72 hours after PGF2α, but were reduced when AI was performed at 48 or 80 hours after PGF2α [42]. The 7-11 Synch protocol improves synchrony of estrus over other protocols (Select-Synch, MGA Select) and peak estrus response typically occurs 56 hours after PGF2α [42]. Pregnancy rates resulting from fixed-time AI after administration of the 7-11 Synch protocol were optimized when AI was performed 60 h after PGF2α. Mean pregnancy rates resulting from fixed-time AI utilizing the MGA Select and 7-11 Synch protocols are reported to be 67 and 61 %, respectively [43]. Protocols based on MGA, however, are directed at beef cattle, to avoid any adverse effects on human health due to the possibility of milk residue if used in dairy cows. Moreover, MGA is also an anabolic agent for growth promotion, its utilization in cattle feed could be banned or utilized under restrict regulation in some countries as in the EU. There are no other reports in the literature on the use of MGA to synchronize estrus in buffaloes except that of Shukla et al., [44]. These workers reported that 22 out of 24 dry buffalo cows fed with MGA showed estrus between Day 4 and 5 after the suspension of MGA feeding, but only 29.1% conceived at the first synchronized estrus while the highest CR (63.6%) was observed at the third estrus post-MGA. These data are in agreement with those found in the bovine by Roussel and Beatty [45] and Patterson et al. [42], confirming low fertility in buffalo cows at the first synchronized estrus, following MGA treatment.

Norgestomet

Norgestomet is a synthetic analogue of progesterone. It is administered as a subcutaneous ear implant containing 3 mg norgestomet, in combination with a single intramuscular injection containing 3 mg norgestomet and 5 mg estradiol valerate (Synchro-Mate B regimen) [46]. The implant is withdrawn after 9 or 10 days. Insemination can be done 48 h (heifers) or 56 h (cows) after implant removal [47,48]. Regimens involving the use of progestogens sometime additionally include the administration of PGF2α two days before implant removal, in order to eliminate any natural CL [49]. In buffaloes, norgestomet associated to a follicle stimulating hormone like Pregnant Mare Serum Gonadotrophin (PMSG), administered at the moment of implant removal, has been shown to induce estrus in anestrus buffaloes (85 vs 0 % in treated and control group, respectively) [50], while the utilization of norgestomet ear implant plus estradiol valerate not associated with a follicle stimulating hormone, resulted in low efficiency in synchronizing ovulation during the non-breeding season [5]. Therefore, PMSG treatment may be an important tool for increasing synchronization when norgestomet is utilized in anestrous buffaloes. Previous studies on the use of fixed time AI at 48h and 72h from implant removal did not result in satisfactory conception rate [51]. More recently, Carvalho et al., [52,53] reported good results in term of synchronization of ovulation and conception rate when norgestomet was associated with PGF2α and eCG at the time of implant removal, followed by an injection of GnRH two days later; fixed time AI in this case was done at 64 h from progestin withdrawal.

As for the MGA, the utilization of the commercial products containing norgestomet is restricted in the EU and some other countries (i.e. Australia and New Zealand) because of the association with the estradiol valerate. Concerns with regards to consumer health, following exposure to steroidal residues in food products, have led to recent changes in EU legislation prohibiting the use of estradiol-17β, and its related ester derivatives, in food-producing animals for the purposes of estrus synchronization, starting from October 2006 [54]. In the USA, estradiol is no longer available for use in food producing animals [55].

Intravaginal drug delivery devices

A number of commercially available intravaginal drug delivery devices, based on both natural progesterone and synthetic progestagen, have been developed with the aim of controlling the estrous cycle in livestock [56]. The most common intravaginal progesterone devices are available in the market by the name of PRID® (Progesterone Releasing Intravaginal Device) and CIDR®(Controlled Internal Drug Releasing device) containing 1.55 g and 1.38 g of progesterone, respectively [6]. When the device is inserted into the vagina, progesterone is slowly released over the treatment period. Under the influence of progesterone, normal pituitary gonadotrophin output is inhibited and the ovarian cycle is interrupted. The removal of the device results in the rapid decline of plasma progesterone and the onset of estrus in animals responding to treatment [57,58].

During the short-term device insertion (7-10 days), in the presence of a CL, progesterone diffusion from the PRID/CIDR/DIB does not affect spontaneous luteolysis, thus to be effective in synchronizing estrus, a luteolytic agent must be incorporated in the synchronization treatment (Fig. 2) [10]. Both estradiol and PGF2α have been used as luteolytic agents in association with progesterone devices. Estradiol has been given at the start of the treatment: intramuscular injection when CIDR was used; vaginal administration as gelatin capsule attached to the inner surface of the coil, when PRID was utilized. The estradiol, when administered immediately after ovulation, seems to act as an antiluteotrophic agent; when it is administered in the presence of an active CL, it is generally luteolytic although its ability to induce regression of the CL is very limited (3-5 days after ovulation) [10]. For this reason and due to the recent restriction in EU regulations concerning the use of estradiol, prostaglandin is the luteolytic agent of choice to incorporate into a progestin treatment.

Figure 2. Estrus synchronization scheme based on simulating CL function: progesterone intravaginal devices. Two fixed-time AI are required because of the variability in ovulation time.

Natural or synthetic progesterone containing devices (injections, intravaginal pessary, ear implants) have been used successfully to improve synchrony of estrus and conception in buffaloes (Table 1). The synchronization protocols, however, are efficient if buffaloes are cyclic and therefore if they are used during the breeding season (autumn). In the spring season, there is a higher variability between the onset of estrus and the time of ovulation and it is harder to establish the correct time for AI.

Baruselli, [38] used a progesterone intravaginal pessary (CIDR-B) or a progestagen ear implant (CRESTAR) along with estradiol to study the follicular dynamics during the period in which the implant was kept inside the vagina, in order to evaluate the appropriate moment for fixed time insemination in buffalo cows. The author found that the CRESTAR protocol was not efficient in synchronizing estrus and ovulation, while animals treated with the CIDR-B protocol ovulated, although the percentage of ovulated animals (66.6%) and synchronization of ovulation (varying from 32 to 96 hours) did not demonstrate a good efficiency. The author showed in previous work that the use of a progesterone pessary (PRID), associated with PMSG and prostaglandin, is able to control ovulation and induce a good rate of synchronization in buffaloes [59]. Using this synchronization treatment schedule during the peak breeding season (autumn) and the low breeding season (spring), we found no differences in the fertility rate between the two seasons considered: in fact the CR was 46.2% and 44.3% in autumn and in spring respectively. Rao and Rao [60] investigating the PRID treatment both during the peak and the low breeding season found that fertility was much higher during October to January (peak breeding season) than during the rest of the year (40.7% vs 25.3%). Singh et al., [61] found that the use of gonadotrophin in addition to the PRID treatment ensures a good ovulatory response during the low breeding season.

In fact, during the summer months, they observed a pregnancy rate of 50% in Indian buffaloes synchronized with PRID + PMSG, which was higher than those observed in their previous work using a treatment with PRID alone (8% to 28%) [62,63]. In previous work, the author also found that the use of PMSG increases the fertility that is related to the doses utilized; in fact CR was 26% in buffaloes in which PRID + 500 IU PMSG were used and 17.5% in buffaloes in which PRID was used without gonadotrophin [64]. In our treatment schedule, the addition of PGF2α to PRID + PMSG was useful to avoid the presence of any functional CL at the time the device was removed, which could delay the synchronization, as reported in cattle [65] and in buffaloes [66,67]. The improvement in ovulation and pregnancy rates with the addition of eCG (equine Chorionic Gonadotrophin) to a progesterone-based protocol, during the non-breeding season, is supported also by the work of Murugavel et al. [68] and Carvalho et al., [69]. These authors reported a CR of 40.6 vs 27.3% and 52.7 vs 39.4%, respectively, when eCG was utilized.

To better define the proper time for AI following a PRID synchronization treatment, our group has evaluated the time to LH peak, after pessary removal, in two different seasons [70,71]. Based on these results, we have suggested that 72 and 96h after PRID removal could be more appropriate times for AI in synchronized buffalo cows during the low breeding season, while 48 and 72 hours could work better in the autumn. Utilizing two AI schedules in Italian Mediterranean buffaloes at 72 and 96 hours during the spring season, we have obtained a CR ranging from 47.8 to 64.5% in different years [72-74]. Sekerden [75], utilizing the same protocol during the low breeding season also obtained a CR at AI of 55.1% in Anatolian buffaloes.

Intravaginal progesterone has been also utilized to induce and synchronize estrus in buffalo heifers. Saini et al., [76], using PRID plus PMSG to induce estrus in non-cycling buffalo heifers, reported that all animals in the treated group expressed estrus while none expressed estrus in the control group. These authors reported that more intense estrus symptoms and better conception rate were obtained when PMSG was used with PRID, as PRID treatment alone failed to induce a fertile estrus. Andurkar and Kadu [77], using a CIDR with PGF2α and PMSG, induced estrus in non-cycling buffaloes (either cows or heifers) and found better fertility with a long-term (12 days) than a short-term (8 days) treatment. The author’s work has also shown that the use of PRID plus PMSG treatment is able to induce fertile estrus in non-cycling heifers and resulted in a higher CR, compared to controls (65 vs 25%)[78]. Using the PRID regime it is possible to synchronize estrus in cycling heifers, overcoming the problem of estrus detection and increasing the effectiveness of AI programs in buffalo heifers. The CR at AI obtained in the author’s work, utilizing either cycling or non-cycling animals was around 37% [78,79]. This was a good result relative to the small amount of data reported in literature (Table 2). Honnappagol and Patil [37], using an analogue of PGF2α to synchronize estrus in cycling Surti buffalo heifers, had a CR to AI ranging from 12.5 to 62.5%. Zicarelli et al., [80], using PRID in Italian cycling buffalo heifers, reported a CR to AI of only 20.2 %; the same group of researchers, using prostaglandin or prostaglandin + GnRH had a CR of 55.0% and 44.4% respectively [35]. Kumaresan and Ansari [81], utilizing AI on spontaneous estrus, reported a CR ranging from 16.67 to 33.33% in relation to the stage of estrus; the highest CR was obtained when heifers were inseminated at 18-24 hours after estrus. The efficiency of PRID in inducing fertile estrus in buffalo heifers and improving CR at AI during the low breeding season was confirmed by a subsequent study from our group [82]. In this last study, the CR obtained with the PRID protocol was higher compared to the one obtained with the protocol utilizing GnRH and PGF2α (39.0 vs 25.9%) This trend was more evident in heifers <24 months of age, showing that the PRID protocol was more effective in inducing estrus in pre-pubertal animals.

Therefore, the use of progesterone associated with PMSG and prostaglandin can be successfully employed to increase the effectiveness of AI programs improving the fertility rate during the low breeding season. Another advantage of progesterone-based treatments is that they can be used to induce estrus in pre-pubertal heifers [78] and anestrus buffalo cows during the post-partum period [4].

Manipulating Follicular Growth and Timing of Ovulation

In the past decades, the use of ultrasonography has helped researchers understand follicular growth patterns, developing protocols that minimize animal handling and ensuring good fertility rates. Estrus control treatments should affect the wave pattern by preventing the development of persistent dominant follicles and the recruitment of the future ovulatory follicle regardless of the stage of the wave at the time of treatment. This would allow synchronous ovulation of a growing dominant follicle [10].

In cattle, it has been shown that synchronization of a follicular wave and subsequent estrus can be achieved by the association of GnRH and prostaglandin [83]. The LH release induced by GnRH injection causes ovulation or luteinization of the dominant follicle (DF) leading to emergence of a new follicular wave. The subsequent injection of prostaglandin causes the regression of the CL. The low progesterone environment stimulates the development of the newly formed DF and estrus and ovulation occur 2-3 day after prostaglandin injection [10,84]. These insights into follicular growth and wave manipulation, led Pursley et al., [83] to develop an estrus synchronization program named Ovsynch. This program has been extremely successful in cattle for insemination at a fixed time without the need for estrus detection, because there is synchronization of ovulation of an ovulatory follicle by administration of GnRH 48h after the prostaglandin injection. The Ovsynch consists of an injection of GnRH (Day 0) followed by an injection of prostaglandin (Day 7) and an additional injection of GnRH 48 h later (Day 9), and fixed time AI is done 16–20h after the second GnRH injection [83] (Fig. 3). For this protocol to succeed there must be a DF present at the time of the first GnRH injection. A follicular wave in recruitment or in early selection phase will not be synchronized because the DF is either not present or is physiologically immature [49,85]. Another limitation of this protocol is that it works better when the animals are cyclic, so it is not recommended in heifers unless they have reached puberty, and it is not recommended in buffaloes during the non-breeding season. The main problems of the Ovsynch protocol in non-cyclic buffalo cows are early and asynchronous ovulations [86,87].

Figure 3. Ovsynch program: estrus synchronization scheme based on manipulating follicular growth and time of ovulation. Usually, only one fixed time AI is done because ovulation is synchronized in a short period of time by the 2nd GnRH injection.

Over the last decades, the efficacy of Ovsynch protocol in the application of AI programs without estrus detection, has been studied on different buffalo breeds in Latin America [38,88-90], in Europe [74,91-93] and in Asia either in River [94,95] or in Swamp buffaloes [96] (Table 3).

The efficacy of the treatment in this species depends mostly on the breeding season [97]. Baruselli, [38] using the Ovsynch protocol had a CR of 48.8% in buffaloes inseminated during the breeding season (autumn-winter) and 6.9% in those that were inseminated during the non-breeding season (spring-summer). Other authors using the Ovsynch protocol reported a CR at AI ranging from 56.5% [88], if used during the breeding season, to 36.0-42.5% [74,91] if used in the period of transition to seasonal anestrus. This difference can be attributed to the presence of a higher rate of non-cyclic animals due to the suboptimal function of the hypothalamic-pituitary-gonadal axis that occurs during spring-summer period in buffalo [98]. In fact, a higher CR is reported in cyclic compared to non-cyclic buffaloes when Ovsynch is used during the spring–summer period [86,87,92].

Efficiency of Hormonal Protocols for Fixed Time AI during the Breeding Season

Use of PGF2α, Progesterone (intravaginal or subcutaneous) or Ovsynch and its modification (i.e. Cosynch, Heatsynch, Presynch, Doublesynch)

Estrus synchronization programs that utilize progesterone or progestagens (i.e. PRID, CIDR, and Norgestomet) are effective in synchronizing estrus in cyclic buffaloes. PGF2α is used during progesterone treatment to induce luteolysis and improve estrus synchrony. Usually buffaloes are treated with progesterone for 10 days and a luteolytic dose of PGF2α is administered 0-3 days before withdrawal [5,6]. Progesterone or progestagen treatments have been applied during the breeding season resulting in a good CR [38], but timed AI cannot be easily used due to the large variability in the treatment-ovulation interval; therefore, when progesterone based synchronization program is utilized; it is advisable to use a double AI at 24 h intervals. In order to synchronize ovulation in a shorter period of time and perform a single timed AI, the treatment should be used in combination with GnRH [99,100].

Hormonal treatment using only PGF2α has been used in buffaloes [27,32,35]. Two injections 11 days apart can synchronize estrus in cyclic animals; however, the large variability in the interval from the last PGF2α injection to estrus and ovulation does not always result in acceptable CR after fixed time AI.

The use of Ovsynch protocols during the breeding season with fixed time insemination 16-20h after the second GnRH injection, have yielded acceptable conception rates (48%-56%) in buffaloes [38,88,96] although lower conception rates (around 35%) have also been reported [90,93,95] (Table 3). The difference seems to depend on the stage of the estrus cycle at the start of the treatment, as the degree of synchronization after Ovsynch in cyclic animals could be improved by initiating treatment in the presence of a DF [92]. Nevertheless, it seems to be the most appropriate protocol for cyclic buffaloes during the breeding season.

Figure 4. Estrus synchronization scheme based on manipulating follicular growth and timing of ovulation: Modification of the Ovsynch program- Heatsynch, Doublesynch, Estradoublesynch- utilized with success in cyclic buffaloes.

In the last years, modifications of the classical Ovsynch protocol have been developed trying to increase reproductive efficiency (i.e. Heatsynch, Cosynch, Presynch, Doublesynch) (Fig. 4). A protocol that substitutes the second GnRH injection with estradiol (Heatsynch) developed recently in cattle, has been successfully used in buffaloes [101]. The Heatsynch protocol leads to a high level of estrus expression and has the advantage of a reduced hormonal cost. The schedule for Heatsynch consists of GnRH + prostaglandin after 7 days + estradiol after 24h and timed AI after 48h. The choice of the protocol depends also on the country regulatory requirements for hormonal treatments; estradiol, for example, is not approved in Europe or in the United States. In order to reduce the number of times animals need to be restrained for treatment purposes, a modification to the Ovsynch protocol, called Cosynch, has been introduced, in which cows are inseminated at the time of the second GnRH injection [102]. The Ovsynch protocol, like the other protocols i.e. Cosynch and Heatsynch, based on follicular wave synchronization, is more successful when the initiation of treatment is between Days 5 to 12 of the estrous cycle. Several strategies employing hormonal treatment before initiating the Ovsynch protocol have been utilized to improve the efficacy of the treatment and for the fixed time AI to be successful. A pre-synchronization strategy, in which two injections of PGF2α at 14 days interval are administered before the initiation of Ovsynch (Pre-Synch) has shown to improve CR in dairy cows [103]. A disadvantage of the Pre-Synch protocol is that a higher number of injections are required and the treatment occurs over a longer period of time.

A new synchronization method that includes the administration of an additional PGF2α injection 48h before beginning the Ovsynch protocol has been recently developed in cattle for both cyclic and anestrus cows [104,105]. This new protocol is named Doublesynch, as it resulted in synchronized ovulation after both the first and second GnRH treatments. The Doublesynch protocol produced efficient synchronization of ovulation also in buffaloes and resulted in satisfactory pregnancy rates in cycling animals irrespective of the stage of the estrous cycle [106]. The effect of a new estrus synchronization protocol, the Estradoublesynch protocol (in which estradiol benzoate replaces the second GnRH injection of the Doublesynch protocol) was recently investigated in buffaloes [107] and was mentioned to be efficient in terms of conception rates in both cycling and anestrus buffaloes during the breeding season.

Efficiency of Hormonal Protocols for Fixed Time AI during the Non-Breeding Season

Use of Intravaginal Progesterone (PRID/CIDR/DIB) Associated with PMSG and PGF2α or with Ovsynch

The use of progesterone-based protocols (PRID or CIDR device) during the non-breeding season allows for the successful use of AI even when buffaloes are in anestrus. These protocols induce higher rate of ovulations than the Ovsynch protocol when buffaloes are non cyclic because of the priming effect on the hypothalamic–pituitary–ovarian axis [98]. In the low breeding season, it is recommended to use gonadotrophin in addition to the progesterone-based protocol, to ensure a good ovulatory response and to enhance the CR [64,69,72]. The variability of the interval between the end of the treatment (device removal) and ovulation is more easily seen following the progesterone-based protocol compared to the Ovsynch protocol; therefore, it is advisable to use double AI at 24 h intervals when using PRID or CIDR device.

Comparing the efficiency of PRID and Ovsynch protocols in buffaloes, with respect to the season, our group found that both treatments showed the same efficiency in obtaining estrus synchronization and good CR at AI in the spring. Although the fertility rate did not differ significantly between the PRID and Ovsynch protocols (47.82% and 42.55% respectively), a higher CR was found in buffaloes synchronized with PRID compared to Ovsynch, as PRID treatment was able to overcome the anestrus status in non-cycling animals [74]. This conclusion was supported by the work of Baruselli et al., [108], who reported that using the Ovsynch protocol they had a CR of 48.8% in buffaloes inseminated during the breeding season (autumn-winter) and 6.9% in those inseminated during the non-breeding season. In fact, the same researchers, comparing CIDR + eCG + hCG treatment to GnRH + PGF2α + GnRH (Ovsynch protocol) during the non-breeding season, found a higher CR at AI in animals treated with CIDR (53.5% vs 28.2%) [109]. The combination between progestagens and hormones that synchronize ovulation has been found to increase CR in bovine [110-112]. Therefore, in order to decrease the variation in ovulation time and increase the effectiveness of fixed time AI when using progesterone-based protocols, the use of GnRH in association with progesterone treatment was evaluated in buffaloes. In previous work, the author utilized an injection of GnRH 16h before the first insemination in buffaloes artificially inseminated at 72 and 96h from PRID removal. According to the protocol utilized in this trial, the use of GnRH in association with PRID did not improve the CR in buffaloes undergoing AI (45.2% and 64.5% with PRID + GnRH or PRID respectively) [73]. Satisfactory results (53.5% CR), during non-breeding season, have been obtained by Baruselli et al., [109] using a progesterone intravaginal device (CIDR) associated with eCG and an injection of hCG (human chorionic gonadotrophin) 14h before AI, since the animals received only one insemination at 62 h from CIDR withdrawal. Similar results (52,7% CR) were reported by Carvalho et al., [69], utilizing GnRH 24h after progesterone device removal and only one AI, 16h after GnRH injection. In recent years, our group has obtained good results in CR (from 45 to 60%) by adopting PRID + PMSG + PGF2α, combined with an injection of GnRH 48h after PRID removal and only one AI 16-20h from GnRH administration [99] (Fig. 5).

Figure 5. Estrus synchronization scheme to increase the efficiency of fixed time AI in buffaloes during the non-breeding season: combination between a progesterone intravaginal device program and GnRH. The administration of GnRH 48h after device removal is effective in synchronizing ovulation and improving conception rate, performing a single fixed time AI.

Figure 6. Estrus synchronization scheme to increase the efficiency of fixed time AI in buffaloes during the non-breeding season: insertion of progesterone intravaginal device during Ovsynch program. Progesterone supplementation, between the first GnRH and the PGF2α injection, improves follicular wave synchrony and the establishment of pregnancy in anestrus buffaloes.

Studies have been done on buffaloes treated with intravaginal progesterone devices during an Ovsynch protocol (Fig. 6). The exogenous progesterone was found to have no positive effects on synchronized ovulation rate or on CR (57.5 vs 55.4%, in the group with progesterone and the group with only Ovsynch respectively), probably because the animals were acyclic [113]. Murugavel et al., [68] reported a CR of 40.6%, in anestrus buffaloes treated with CIDR inserts during an Ovsynch protocol and inseminated at natural estrus.

To evaluate the effect of different circulating progesterone concentrations during an ovulation synchronization protocol, based on an intravaginal progesterone protocol for fixed timed AI in buffalo cows, a reused progesterone device treatment was considered. The results from these studies, provided evidence that the lower circulating progesterone concentrations released from reused devices is sufficient to control the ovarian follicular growth without detrimental effects on pregnancy responses in seasonal anestrus buffalo cows subjected to an ovulation synchronization protocol for timed AI [114]. Regardless of the treatment with a new or a previously used progesterone device, satisfactory ovarian responses were achieved also when buffalo cows were subjected to progesterone based protocols during the breeding season [100].