The plasma P4
concentrations, diameter (Mean±SEM) of CL and DF on different days of intervention and comparative analysis of these parameters based on subsequent pregnancy status of group-I (Presynch-Heatsynch) and group-II (Heatsynch group) have been presented in (Tables 1 and 2), respectively.
Table 1: Different parameters in relation to pregnancy in buffaloes subjected to Presynch-Heatsynch protocol.
Effect on ovarian parameters
Dominant follicle diameter
Table 2: Different parameters in relation to pregnancy in buffaloes subjected to Heatsynch treatment.
The diameter of DF on day -2 as well as on day 0 remained larger (P<0.05) in buffaloes of group-I than of group-II (Fig 3). Furthermore, DF diameter on the day of EB injection (P<0.05) and FTAI (P<0.01) in group-I remained higher compared to group-II buffaloes. Within each group, size of DF during second follicular wave (that emerged after induction of ovulation by GnRH injection on day 0) increased (P<0.05) by the time of FTAI after PGF2
α on day 7 in both groups (Group-I: 10.21±0.25 to 15.04±0.35; Group-II: 10.01±0.29 to 12.81±0.31 mm, respectively).
However, in group-I, DF diameter was larger (P<0.05) on the day of first GnRH injection in buffaloes that became pregnant compared to non-pregnant buffaloes i
. 9.54±0.35 versus (vs.) 8.18±0.53 mm, respectively. Likewise, DF diameter on rest of the days (Day 7, 8 and 10) during ovulatory wave remained larger (P<0.01) in buffaloes that became pregnant than non-pregnant (Table 1). Similarly in group-II, DF diameter was larger on the day 0 (P<0.05), days 8 and 10 (P<0.01) in pregnant compared to non-pregnant buffaloes (Table 2).
Corpus luteum diameter
The diameter of CL on commencement of the study (day14; 9.66±0.6 vs. 8.38±0.54 mm) did not differ (P>0.05) between the groups. However, CL diameter differed (P<0.01) between the two groups on days-2, 0 and 7. Diameter of CL decreased abruptly (P<0.05) after PGF2
α injection on day-2 (Group-I) and day 7 (Group-II) (Fig 2).
Fig 2: Diameter of corpus luteum (mm) and progesterone concentration (ng/ml) in buffaloes.
In Group-I, CL diameter remained significantly (P<0.01) larger in pregnant buffaloes on all the days of three PGF2
α injections compared to their non-pregnant counterparts (Table 1). In trend to the serum P4
profiles, CL diameter decreased abruptly (P<0.05) in both pregnant and non-pregnant buffaloes following luteolysis caused by PGF2
α administration on day 7 of the treatment.
The diameter of CL remained comparatively greater in group-II on day-14 (Table 2). The CL size decreased abruptly (P<0.05) following PGF2
α injection on day 7 of the treatment.
Effect on plasma steroid hormone profiles
The mean plasma E2
concentrations on first day (day-14) of the treatment were similar in both the groups. However, E2
concentrations on days -2, 0 and 10 were significantly higher in group-I compared to -II (Fig 3). After PGF2
α administration on day 7 in both groups, E2
concentration increased (P<0.05) on the day of EB injection, which further increased (P<0.05) on the day of FTAI. The E2
profiles, in both groups, were comparable between the pregnant and non-pregnant buffaloes; however, the concentrations remained higher (P<0.05) on the day of EB injection in pregnant compared to non-pregnant buffaloes (Table 1 and 2).
Fig 3: Diameter of dominant follicle (mm) and estradiol concentration (ng/ml) in buffaloes.
concentration remained similar (P>0.05) in both the groups on first day (day -14) of the experiment (Fig 2). However, P4
concentration between the two groups differed significantly (P<0.01) on days-2, 0 and 7. On the day of AI (day 10) P4
concentration remained non-significantly higher in group-II. In Group-I, PGF2
α administration on day -14 seems to have caused luteolysis and thus, assisted in ovulation of the DF four-five days later. The second PG shot in this group (on day-2) resulted in the luteolysis of CL formed in response to ovulation of the previous follicular wave.
Unlike group-II buffaloes, luteolysis by 2nd
PG administration on day-2 was indicated in group-I by significant drop (P<0.05) in plasma P4
concentration, on day -2 compared to the day 0 (1.80±0.12 vs. 0.50±0.04 ng/ml).
In Group-I, P4
levels remained higher (P<0.01) on days of all three PGF2
α injections in buffaloes that became pregnant compared to those who failed to conceive (Table 1). In both pregnant and non-pregnant buffaloes, P4 levels decreased abruptly (P<0.05) after luteolysis caused by PGF2
α administration on day 7 of the treatment. However, on the day of FTAI, P4
concentration was higher (P<0.01) in non-pregnant buffaloes than those that became pregnant. Similarly in pregnant buffaloes of Heatsynch group, P4
levels remained higher on all days of treatment except on the day of FTAI as compared to non-pregnant ones (Table 2). However, on the day of FTAI, P4
concentration remained higher (P<0.01) in non-pregnant compared to pregnant buffaloes. In pregnant buffaloes, P4
concentration decreased (P<0.05) following injection of PGF2
α on day 7, unlike in non-pregnant buffaloes. On the day of FTAI, P4
concentration did not decrease from the day of PGF2
α administration in buffaloes that failed to become pregnant. High P4
levels on the day of FTAI (Day 10) did not seem to affect ovulatory response as more than 90% animals in both groups ovulated post-AI (Fig 2).
Effect on fertility parameters
In the present study, an ovulatory response of 93.33% (28 out of 30 buffaloes) and 90.00% (27 out of 30 buffaloes) was observed in Group-I and -II, respectively (Fig 2). The conception rate in the present study was significantly higher (P<0.05) with Presynch-Heatsynch than the Heatsynch treatment (66.66 vs. 40.00%, respectively).
Poor reproductive performance, manifested by long calving intervals, could result in huge economic losses due to low milk yield, high replacement costs and culling rates in buffaloes. Estrus synchronization was considered as an effective tool to enhance submission and pregnancy rates in buffaloes. It eliminated or atleast reduced the problem of estrus detection which was considered as the major limitation in reproductive management of buffaloes. Ovsynch protocol was developed to allow timed artificial insemination (TAI) without the need for detection of estrus (Pursley et al., 1995).
Later, Ovsynch protocol was also applied to buffaloes (Ghuman et al., 2008).
However, during the last few years another promising estrus synchronization protocol called Heatsynch was developed, that incorporated combination of GnRH-PGF2
α-EB injections followed by FTAI. The EB was a less expensive hormone in place of second GnRH injection of Ovsynch protocol. The major advantages of Heatsynch protocol were reduced hormone costs, increased estrus intensity, ease in scheduling and implementation, since all injections and AI are at 24 and 48 h intervals (Mohan and Prakash et al., 2014).
Presynchronization of estrus prior to incorporation of Ovsynch program was reported to increase pregnancy rates by 10-12% in bovines (Moreira et al., 2001; Navanukraw et al., 2004).
Moreover, presynchronization could be incorporated during the voluntary waiting period, thereby eliminating the delay in the first postpartum service. Therefore, incorporation of presynchronization prior to Heatsynch might prove beneficial in buffaloes. The conventional method of presynchronization involved administration of two PGF2α injections 11 to 14 days apart, with the last injection given 14 days before initiation of any GnRH based breeding protocol (Moreira et al., 2000).
However, in the current study, a modified presynchronization was adopted before initiation of Heatsynch (Presynch-Heatsynch) protocol that involved administration of two PGF2
α injections at 12 days apart, last PGF2
α injection given 2 days prior to the first GnRH of Heatsynch protocol was investigated in 60 lactating buffaloes with the aim of developing a shortened protocol.
In the present study, favourable effect of additional presynchronization was indicated by larger diameter of DF on day-2 as well as on day 0. Administration of PGF2
α before first GnRH injection in any of the GnRH based protocol enhanced the pituitary release of LH in response to GnRH (Mirmahmoudi et al., 2014).
It had been established for GnRH based protocols that animals ovulating after first GnRH injection are more likely to have functional DF capable of ovulation after final GnRH injection (Vasconcelos et al., 1999).
Larger diameter of DF during ovulatory wave in pregnant than in non-pregnant buffaloes of the present study indicated that in pregnant buffaloes, first GnRH possibly ovulated the existing follicle of larger size resulting in functional DF capable of ovulation after injection of EB on day 8 of the protocol. Hence, the better ovulation induction after GnRH administration could be one of the most important reasons for the greater fertility in pregnant animals of both the groups. These observations were in corroboration with the earlier report of peak E2
concentration (of 35.8 pg/ml) one day prior to estrus in bovines (Bachalaus et al., 1979).
In buffaloes, E2
concentration at the time of estrus increases upto 45-50 pg/ml (Batra and Pandey, 1983)
. The E2
concentrations were positively related to the size of follicle and as bigger sized follicles secreted more E2
compared to smaller ones (Palta et al., 1998).
This relationship was also consistent in the present study.
The sufficient P4
concentrations are pre-requisite for proper growth of the ovulatory follicle (Bisinotto et al., 2010
and Wiltbank et al., 2011).
It was believed that effect of P4
on fertility involved a change in the pattern of LH release. The changes in LH pulse frequency were linked with altered follicular maturation and subsequent embryo survival (Cerri et al., 2011).
Several earlier reports emphasized the importance of high concentration of P4
during the follicular growth of pre-ovulatory DF for successful establishment of pregnancy (Meisterling and Dailey, 1987; Bilal et al., 2016).
Previous studies have indicated that the P4
concentrations during the development of DF influenced the fertility since DFs of first and second follicular wave grow under different P4
environments (Denicol et al., 2012).
The first wave DF grows under sub-luteal phase P4
level (<1.5 ng/ml) for a few days followed by luteal phase level, whereas DF of ovulatory wave grows under luteal phase level (>2 ng/ml) of P4
, prior to luteolysis (Sartori et al., 2004
and Denicol et al., 2012).
The ovulation following PG administration occurred at 60-156 h in bovines (Brito et al., 2002).
Thus, administration of the GnRH on day 0 could have facilitated the ovulation of the follicular wave DF that was initiated after ovulation at around day 10. Administration of the third PG on day 7 resulted in luteolysis, whereas injection of EB on day 8 resulted in LH peak required for subsequent ovulation of the ovulatory follicle with better expression of estrus.
Administration of GnRH on day 0 in group-I animals was able to induce ovulation of DF and formation of CL as indicated by higher (P<0.05) P4
concentration on day 7 (2.61±0.18 ng/ml) than on day 0 and 8 (0.50±0.04 and 0.94±0.80 ng/ml, respectively).
In group-II of present study, P4
concentrations on day 0 and 7 were similar due to the fact that no PGF2
α was administered prior to first GnRH injection. However, after luteolysis, the P4
concentration decreased abruptly (P<0.05) after day 8. Similarly in group-I, P4
levels reduced (P<0.05) after day 7 till day of FTAI. The above findings indicated that pre-synchronization before start of Heatsynch protocol resulted in low P4
environment before first GnRH injection (day 0) leading to ovulation in most of the buffaloes post GnRH injection. This might have further led to optimum DF size at days of EB injection and FTAI for ovulation and pregnancy to occur. The significantly higher P4
levels in buffaloes that became pregnant compared to those that failed to become pregnant in the present study, clearly indicated that ovulatory follicle developed well under higher P4
environment in pregnant buffaloes of both groups. This finding corroborated with the earlier reports of Cerri et al., (2011).
level throughout ovulatory follicle development was related with increased intra follicular levels of IGF-1 and improved pregnancy rates (Wiltbank et al., 2011).
during growth of ovulatory follicle may increase pregnancy rates by atleast 10 per cent in bovines after FTAI (Bisinotto et al., 2010).
Another important finding of the present study was presence of supra-basal levels of P4
at the time of FTAI in buffaloes that failed to conceive in both groups. High P4
concentration near AI might reduce pregnancy rate by altering gamete transport through compromised oviductal or uterine contractility, thereby, reducing the fertilization rates (Hunter, 2005). The conception rate obtained in the present study was higher than those reported by Mohan et al., (2009)
and Mirmahmoudi et al., (2014)
in buffaloes subjected to Heatsynch protocol (32.5 and 33.33%, respectively). Further, conception rates obtained in Presynch-Heatsynch group (66.66%) was higher than reported in earlier studies comparing conception rate following AI on second wave ovulation in buffaloes (Hoque et al., 2014)
and dairy cows (Sartori et al., 2009)
(44.4 and 48.0%, respectively).