Artificial Insemination at Fixed Time in Bufalloes

To maintain a calving interval of 13-14 month in buffaloes, successful breeding must take place within 85-115 days (d) after calving. Complete uterine involution and resumption of ovarian activity and heat expression usually takes place around 20-50 d post partum (dpp); therefore, there is a window of 35-95 d to rebreed a cow and get her pregnant to maintain the desired calving interval. Although artificial insemination (AI) has the potential to make a significant contribution to genetic improvement in buffaloes, its practical application has been difficult because poor estrus expression by cows and poor estrus detection by humans, a variable duration of estrus and the difficulty to predict time of ovulation. More recently, the development of protocols for synchronization of ovulation and fixed timed insemination (TAI) in buffaloes have been used to overcome these constrains and be able to use more extensively AI in commercial herds. Nevertheless, resynchronization of ovulation and TAI still remains a problem herds managed under extensive conditions for similar reasons abovementioned. Very recently, we did four field trials to study the efficacy of different protocols that combined use of GnRH, or estradiol benzoate (EB), prostaglandin (PGF) and intravaginal progesterone (P4) releasing device (PIVD) or norgestomet ear implant (NOR) to resynchronize estrus and ovulation at day 18 post AI in buffalo cows under commercial conditions.


Introduction
To maintain a calving interval of 13-14 month in buffaloes, successful breeding must take place within 85-115 days (d) after calving. Complete uterine involution and resumption of ovarian activity and heat expression usually takes place around 20-50 d post partum (dpp); therefore, there is a window of 35-95 d to rebreed a cow and get her pregnant to maintain the desired calving interval. Although artificial insemination (AI) has the potential to make a significant contribution to genetic improvement in buffaloes, its practical application has been difficult because poor estrus expression by cows and poor estrus detection by humans, a variable duration of estrus and the difficulty to predict time of ovulation. More recently, the development of protocols for synchronization of ovulation and fixed timed insemination (TAI) in buffaloes have been used to overcome these constrains and be able to use more extensively AI in commercial herds. Nevertheless, resynchronization of ovulation and TAI still remains a problem herds managed under extensive conditions for similar reasons abovementioned. Very recently, we did four field trials to study the efficacy of different protocols that combined use of GnRH, or estradiol benzoate (EB), prostaglandin (PGF) and intravaginal progesterone (P 4 ) releasing device (PIVD) or norgestomet ear implant (NOR) to resynchronize estrus and ovulation at day 18 post AI in buffalo cows under commercial conditions.

First trial
In the first field trial, we assessed with ultrasonography the ovarian follicular dynamics to study the efficacy of a combined treatment of GnRH, PGF and NOR to synchronize and resynchronize ovulations in TAI programs. Eighteen Mediterranean buffalo cows with a body condition score (BCS) of 2.70±0.26 (scale 1-5) from a farm in northeastern Corrientes Argentina (27 • 20' 33" S and 58 • 08' 27" W) were used in the study. Cows were randomly assigned to one of 3 treatments (TRT, Figure 1 Figure 1). Daily ultrasounds and blood samples were taken from day -3 to day 2 during synchronization and from day 18 to day 30 during resynchronization ( Figure 1). Blood samples were stored at -20 C • until P4 concentrations were analyzed by RIA (Count-A-Count®, DPC, Los Angeles, USA; intra-assay CV, 3.78%; Inter-assay CV, 9.28%).

Results and discussion
Dominant follicle diameter prior to ovulation tended to be bigger in TRT1 compared to the TRT2 and TRT3 (12.58±0.67 vs. 10.97±0.74 mm; P<0.07), and it was bigger in resynchronization compared to synchronization (12.56±0.46 vs. 10.70±0.51 mm; P<0.02). On the contrary, even though the diameter of subordinate follicle was bigger with TRT3 compared to TRT1 and TRT2 (5.73±0.45 vs. 4.18±0.43 mm; P<0.02), the diameter of the subordinate follicle was of equal size during synchronization and resynchronization (4.70±0.35 mm). During synchronization, dominant follicle, subordinate follicle, and dominance daily growth rate was 0.55 mm/d, 0.25 mm/d and 0.75 mm/d respectively (Figure 2). During resynchronization, dominant follicle and dominance growth rate changed with a different pattern between treatments (Figure 3). Dominant follicle and dominance growth rate was bigger in TRT1 and TRT2 compared to TRT3 alone (0.87 mm/d and 0.81 mm/d vs. 0.68 mm/d; 0.65 and 0.68 mm/d vs. 0.40 mm/d; respectively; P<0.01; Figure 3A and C). In addition, during resynchronization, subordinate follicle diameter tended to increase continuously for the TRT3, whereas tended to increase and then to decrease with the other two treatments (P<0.09; Figure 3B). Even though the interval from PGF injection to ovulation was longer for TRT1 compared to the TRT2 and TRT3 groups (112.16±7.30 vs. 85.16±8.16 mm; P<0.03), the interval GnRH-ovulation was equal for TRT2 and TRT3 groups (36.54±5.36 vs. 37.83±5.73 mm; P>0.37). During resynchronization, a new wave started and divergence took place at day 19 and 22 for TRT1, at day 20 and 22 for TRT2, and at day 21 for TRT3. TRT2 treatment tended to be more effective in inducing follicle turnover compared to TRT1 and TRT3 alone (100% vs. 81%; P<0.07; (Figure 3). During resynchronization, more dominant follicles ovulated compared to synchronization (100% vs. 81%; P<0.04). Lastly, even though if all 3 treatments were equally efficacious to produce follicle turnover in 90 % of cows, that efficiency tended to be higher during synchronization compared to resynchronization (100% vs. 75%; P<0.07). The diameter and growth rate of the DF reported in our study agree with those reported previously by Presicce et al., (2004). They reported that in pluriparous cows, DF diameter in the first wave was 13.3±0.5 mm and for the second wave was 13.8±0.6 mm and the growth rate was 1.6±0.1 and 1.3±0.1 mm respectively. Similar results were reported very recently by Barkawi et al., (2009). In their study DF diameter for cows with 2 waves was 13 and 15 mm and for cows with 3 waves was 11, 10, and 14 mm. In our study, the DF diameter during synchronization was similar to cows with 3 waves and during resynchronization with cows of 2 waves of their study. Furthermore, the DF growth rate reported in our study is quite similar to that reported by Awasthi et al., (2007). In their study, cows with normal estrus had similar diameter and growth rate compared to our cows during synchronization but was smaller compared to our cows during resynchronization. Progesterone concentrations previous to PGF reported by us in this study are higher than those reported previously by Chauman et al., (1983) and by Kumar et al., (1991). Maybe these higher P 4 concentrations reported here are responsible for lower growth rate of DF prior to PGF administration when compared to growth rates reported previously by others (Presicce et al., 2003(Presicce et al., , 2004Awasthi et al., 2006Awasthi et al., , 2007Barkawi et al., 2009).

Results and discussion
At synchronization, the percentage of cows AI was lower for the HDAI group compared to the TAI groups (80% vs. 100%, P<0.01; Table 1). On the contrary, the synchronization pregnancy rate (33%), the of cow AI (97%) and percentage of cows pregnant at resynchronization (31%), the cumulative pregnancy rate for both AI (56%), the pregnancy rate for natural service (50%) and final cumulative pregnancy rate (78%) were similar between treatment groups.

Material and methods Third and four trial
Lastly, in the third and forth field trials, we assessed the fertility obtained with a combination of GnRH, PGF and PIVD or EB, PGF and PIVD were used to synchronize and resynchronize ovulation in TAI programs in two commercial farms.
In the third field trial, 81 Mediterranean buffalo cows with a BCS of 3.79±0.27 (scale 1-5) from a farm in northeastern Corrientes Argentina (27 • 20' 33" S and 58 • 08' 27" W) were used in the study. Cows were randomly assigned to one of 2 TRT (Figure 5) Table 2). Synchronization pregnancy rate was higher in TRT2 group compared to TRT1 group (68% vs. 44%, P<0.03). However, resynchronization pregnancy rate (78%), percent of embryonic and fetal losses (12%), less and similar result, reports by Vale et al 1989, Campanile et al 2005. The final cumulative pregnancy rate without and with embryonic and fetal losses (93% and 85%) were similar between treatments.
In the forth field trial, 119 Mediterranean buffalo cows with a BCS of 3.17±0.11 (scale 1-5) from a farm in northeastern Corrientes Argentina (29 • 42' 20" S and 59 • 23' 17" W) were used in the study. Cows were randomly assigned to one of 4 TRT (Figure 6)

Results and discussion
Only 104 cows finished the experiment (Table 3). Even though the synchronization pregnancy rate was similar between treatments (41%), more cows were resynchronized with the TAI protocols than with the HDAI protocol (100% vs. 67%, P<0.01). On the contrary, resynchronization pregnancy rate (57%), pregnancy rate to AI (76%), natural service pregnancy rate (30%), and final cumulative pregnancy rate (85%) were similar between treatments (P>0.13). De Araujo Berber et al., (2002) and Ronci and De Rensis (2005) using a GnRH + PGF + GnRH + TAI protocol (Ovsynch) obtained higher pregnancy rates than those achieve by us in these field trials. The findings could be explained because they used weaned cows and most likely all were cycling. Conversely, Paul and Prakash (2005) and Warriach et al., (2008) reported lower pregnancy rates using an Ovsynch protocol. When De Rensis and  supplemented the Ovsynch protocol with P 4 , pregnancy rates were similar to those obtained in our Ovsynch protocols that were supplemented with P 4 . Presicce et al., (2005) using a protocol that combined a PIVD with EB and PMSG obtained higher pregnancy rates compared with an Ovsynch protocol alone, but this higher pregnancy rate is more likely due to the use of PMSG than EB.    Table 3. Reproductive efficiency using two protocols for synchronization and resynchronization of estrus and ovulation in Mediterranean buffaloes.

Conclusion
We can conclude from this series of field trials that the combination of GnRH, PGF and P 4 IVD or EB, PGF and P 4 IVD proved to be efficacious to synchronize and resynchronize ovulation in unweaned buffalo cows. Results from this work, show that a 75% pregnancy rate can be achieved during the first 28 days of the breeding season without heat detection and already taking into account early embryonic and fetal losses. Lastly, it is worth to point out that pregnancy rate achieved in all experiments with TAI protocols was numerically higher than that achieved with HDAI; hence these results indicate that TAI may be a very promising tool for genetic improvement in buffalo herds.