Presynch-Heatsynch: A New Approach to Improve Ovarian and Fertility Responses in Water Buffalo (Bubalus bubalis)

Buffalo is the backbone of dairy industry in India that contributes >55% to the total national milk production. Low reproductive efficiency is considered as a major constraint in dairy buffalo farming. Untimely artificial insemination (AI) leading to decline in conception rate and increase in the calving interval is a major problem in progress of the buffalo dairy industry. Application of estrus synchronization treatments in buffalo provides workable strategy to increase their productive period as these regimens eliminate the problem of estrus detection. A number of these protocols employed in buffalo had achieved partial success with variable results (Ghuman et al., 2008).

Heatsynch protocol has been developed that makes use of a combination of GnRH-PGF2α-estradiol benzoate injection followed by FTAI (Fernandes et al., 2001; Pancarci et al., 2002; Mohan and Prakash et al., 2014). The major advantages of Heatsynch protocol are reduced hormone costs, increased estrus intensity, and easy in scheduling and implementation. Certain limitations of Ovsynch and Heatsynch protocols in buffaloes are reduced pregnancy rates ranging from 20 to 40% were reported in earlier studies (Mohan and Prakash et al., 2014; Singh et al., 2017). It is well established that ovulation of follicle in response to first GnRH injection of any GnRH-based program is a prerequisite for its success (Thatcher et al., 2002 and Cirit et al., 2007). However, at certain stages of the estrus cycle, first GnRH dose of Ovsynch/Heatsynch treatment failed to ovulate the follicle leading to reduced conception (Geary et al., 2000; Ozturk et al., 2010; Mirmahmoundi et al., 2014).

Presynchronization of estrus prior to incorporation of actual GnRH based program may increase pregnancy rates in bovines (Moreira et al., 2001; El-Zarkouny et al., 2004 and Navanukraw et al., 2004). The conventional method of presynchronization involves administration of two PGF₂α injections at 11 to 14 days apart, with the last injection given 14 days before initiation of any GnRH based breeding protocol (Moreira et al., 2000). The major draw back of this method is long duration of protocol and subsequently prolonged service period and the calving interval. Hence, development of short duration pesynchronization method is warranted. However, presynchronization of estrus with double PGF₂α injections before Heatsynch (Presynch-Heatsynch) has neither been reported in buffaloes nor in cattle. The evaluation of the economical alternative protocols to improve the conception in buffaloes and clear doubts associated with fixed time AI (Singh et al., 2006) are warranted. Hence, it was hypothesized that the presynchronization would lead to enhanced submission rate with shorter calving intervals. Thus, the present study was taken up to evaluate the ovarian activity and fertility following modified Heatsynch protocol by addition of two PGF₂α injections (12 days apart, last PGF₂α injection given two days prior to first GnRH) to shorten the duration of the protocol.

Experimental animal
 
The experiment was conducted in 2016-17 on  post-partum (> 60 days in milk) cyclic buffaloes (Bubalus bubalis) maintained at dairy farm of Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India. Buffaloes with history of any pre- and post-partum reproductive and or metabolic issues were excluded. The selected buffaloes had body condition score of 2.5 to 3.5 (Edmondson et al., 1989). Cyclicity of buffaloes was confirmed before enrolling into the experiment by transrectal ultrasonography. The buffaloes were managed in a semi-intensive loose housing system and identified with ear tags.
 
Experimental design
 
The sixty buffaloes enrolled were randomly allocated into two groups and were subjected to experimental protocol as depicted in Fig 1 (Group-I, Presynch-Heatsynch group, n=30; Group-II, Heatsynch group, n=30). Real time B-mode ultrasonography using 7.5 MHz frequency trans-rectal transducer (Z5, Shenzhen Mindray Biomedical Electronics Co. Ltd., Germany) was carried out to record the number and diameter of dominant follicle (DF) and corpus luteum (CL).



Plasma was harvested by centrifugation (3000 rpm, 15 min) from the blood samples taken by jugular venipucture and stored in aliquots at -20°C till further progesterone (P₄) and estradiol (E₂) estimation by Enzyme Immunoassay kits (Arbor Assays, Michigan, USA). This study was duly approved by the Institutional Animal Ethics Committee.
 
Statistical analysis
 
The Chi-square test was employed to compare ovulation and conception rates. Data pertaining to P₄, E₂, DF and CL profiles was analyzed using Unpaired t-test to compare between groups and One way ANOVA to evaluate the effect within the group.

The plasma P₄ and E₂ 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.



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Effect on ovarian parameters
Dominant follicle diameter
 
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 PGF₂α 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.e. 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 PGF₂α injection on day-2 (Group-I) and day 7 (Group-II) (Fig 2).



In Group-I, CL diameter remained significantly (P<0.01) larger in pregnant buffaloes on all the days of three PGF₂α 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 PGF₂α 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 PGF₂α injection on day 7 of the treatment.
 
Effect on plasma steroid hormone profiles
Estradiol concentrations
 
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 PGF₂α 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).


 
Progesterone concentrations
 
Plasma P₄ 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, PGF₂α 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 2^nd 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 PGF₂α 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 PGF₂α 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 PGF₂α on day 7, unlike in non-pregnant buffaloes. On the day of FTAI, P₄ concentration did not decrease from the day of PGF₂α 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-PGF₂α-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 PGF₂α injections at 12 days apart, last PGF₂α 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 PGF₂α 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 PGF₂α 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). Increased P₄ level throughout ovulatory follicle development was related with increased intra follicular levels of IGF-1 and improved pregnancy rates (Wiltbank et al., 2011). High P₄ 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).

In conclusion, the present results indicated that additional presynchronization in Heatsynch protocol might successfully enhance the reproductive efficiency of buffaloes. Moreover, the modified protocol is comparatively of shorter duration (25 vs. approximately 37 days) than the conventional method and might aid to reduced service period and subsequent calving interval in buffaloes.