Blood sampling

Median caudal vein was punctured for collection of blood samples (10 mL) with vacutainers (BD, Franklin Lakes, NJ, USA) at d30 and d60 post FTAI from all cows. Soon after collection, the blood sample was centrifuged @ 2800X g for 20 min and harvested plasma was labeled according to individual cow identification and stored at -20°C till P4 analysis with a solid-phase, radioimmunoassay kit (Coat-a-Count Progesterone, Diagnostic Products Corporation, Los Angeles, CA).

Statistical analysis

Data were analyzed using SPSS Statistics 21.0 for Windows (SPSS Inc. Illinois, USA). Data are expressed as Mean±SEM (Steel et al., 1997). Effect of treatments on PR (in percentage) was calculated by using Chi-square procedure. Differences were considered to be statistically significant at α=0.05. Both OFD and Progesterone concentrations were subjected to one way ANOVA. To see variations among treatments, DMRT was applied. P<0.05 was considered to be statistically significant.

Results

The major aim of the present study was to evaluate the effect of progesterone supplementation during standard Ovsynch protocol for different durations on PR in cyclic lactating cows. Progesterone concentration was also determined at d30 and d60 post AI in all three groups. Pregnancy losses were diagnosed by transrectal ultrasonography on d60 and d90 post FTAI as shown in Table I.

On d30 post FTAI pregnancy rates in OVP0, OVP5 and OVP7 groups were 39.7, 45.5 and 42.6%, respectively. Similarly, on d60 post FTAI, the PR in corresponding groups was 36.2, 43.6 and 37.0%, respectively, while these corresponding values on d90 post FTAI were 32.8, 41.8 and 37.0%, respectively (Table I). Pregnancy losses upto d60 and d90 post FTAI in these groups were 8.7 vs. 9.5, 4.0 vs. 4.2 and 13.0 vs.0.0%, respectively, while overall pregnancy loss upto d90 post FTAI in these groups were 17.0, 8.0 and 13.0%, respectively (Table I). Size of preovulatory follicles of cows in all the treatment groups was also measured through ultrasonography and it was found to be 15.19±0.17, 15.30±0.21 and 15.24±0.19 mm (Mean±SEM), respectively (Table II). Distribution of OFD has been shown in Figure 2. Although the values of

Table I.- Effect of different synchronization protocols on pregnancy rate (%) in lactating dairy cows.

all these variables in OVP0, OVP5 and OVP7 groups differed numerically, statistically there was no significant (P>0.05) difference among all three treatment groups (Tables I, II).

Table II.- Effect of different synchronization protocols on ovulatory follicle diameter (OFD; Mean ± SE) in Lactating dairy cows.

Pregnancy diagnosis was done with ultrasonography. On the basis of pregnancy outcome, animals were divided into either pregnant or non-pregnant group. Progesterone (P4) concentration in all treatment groups was measured on d30 and d60 post AI. The values of P4 concentrations were significantly (P<0.05) lower in OVP0 (6.52±0.32 ng/mL) as compared to those of OVP5 (7.75±0.38 ng/mL) and OVP7 (7.58±0.26 ng/mL) groups, respectively on d30 post FTAI but values between the latter two groups differed non-significantly (P > 0.05).

On the other hand, progesterone concentration differed non-significantly (P > 0.05) among all treatment groups at d60 post FTAI. These values in OVP0, OVP5 and OVP7 groups were 6.37±0.49, 6.75±0.36 and 6.80±0.41 ng/mL, respectively (Table III).

Table III.- Plasma P4 concentrations (ng/mL; Mean ± SEM) in pregnant cows of different synchronization treatment groups on day 30 and 60 post FTAI.

a,b, different superscripts in same row indicate significant differences in the same column (P < 0.05).

Discussion

Ovsynch is one of the most acceptable FTAI estrus synchronization protocol for lactating dairy cows with erratic results (Pursley et al., 1997). The core objective of the current project was to appraise P4 supplementation in addition to standard Ovsynch protocol for 5 or 7 days in comparison with OVP0 in lactating cyclic Holstein-Frisian cows. It is well established fact that supplemented progesterone has a reflective effect on follicular development, LH pulsatility and finally, ovulatory response in dairy cattle. A positive relationship has been documented between P4 profile during follicular development prior to AI and the succeeding PR, suggesting that P4 profile is critical for fertility (Folman et al., 1990). Studies with P4 supplementation have resulted in tight ovulation synchrony in prepubertal heifers and anestrus dairy cows as well. On the other hand, progesterone releasing intravaginal device (PRID) insertion in luteal phase has been found to enhance PR in cattle with low plasma P4 profile as compared to those having elevated P4 profile during estrus (Folman et al., 1990).

In present study, all the cattle were cyclic at the beginning of the experiment. The reason for selection criteria of cyclic animals in present study was based on previous findings of Bisinotto et al. (2013) indicating that 90-95% of cows in diestrus phase of cycle at the introduction of FTAI protocol are anticipated to have CL at the time of PGF2α inj. Contrary to those without CL have 75% chances for the presence of CL at PGF2α. In present study, the pregnancy rates at d30, d60 and d90 post FTAI in OVP5 group of cows were non-significantly (P>0.05) but numerically higher as compared to those of OVP0 and OVP7 groups. Although, Melendez et al. (2006) has observed that CIDR inclusion to FTAI programs has resulted in an increased PR but the cows under treatment were presynchronized with two shots of PGF2α. Circulatory P4 profile has profound impact on pregnancy establishment and its maintenance as well. Its subnormal profile could be a major risk factor which necessitates exogenous P4 supplementation in lactating dairy cattle. Stevenson et al. (2006) evaluated 634 dairy cows for Ovynch+P4 supplementation at 6 different geographical locations in the USA and observed that overall PR was non-significantly (P>0.05) improved in CIDR supplemented group as compared to Ovsynch at day 28 (50 vs. 40%) and day 56 (38 % vs. 33 %), respectively.

Similarly, Kawate et al. (2007) also reported improved PR (58 vs. 50%) in Ovsynch+CIDR treated beef cows as compared to those of single PGF2α treated group. In a similar study, Peeler et al. (2004) recorded higher PR in CIDR inserted cows as compared to FTAI protocol using Estradiol Cypionate, PGF2α and GnRH. Without active CL, supplementation with P4 has resulted in reduced fertility due to persistent follicle development leading to aged oocyte resulting in poor PR after estrus synchronization. They also reported that cattle with elevated P4 profile at the start of standard Ovsynch protocol had an optimal PR (43%) in comparison with other group having low circulatory P4 profile (31%) and anovular group (30%). In another experiment, the cows were subjected to presynchronization with two shots of PGF2α at 14-days interval and breeding protocol was started on either 3rd or 10th day of PGF2α. This controlled breeding protocol was resulted in ovulatory response either from 1st or 2nd follicular wave at FTAI. Higher PR (42%) and P4 profile was observed in cows ovulated during 2nd follicular wave as compared to those with low P4 profile (30%) ovulated during 1st follicular wave (Bisinotto et al., 2013). In another study Cerri et al. (2011) documented that cattle with low P4 profile were found to have higher LH concentration and resulted in not only altered follicular pattern but also uterine origin PGF2α production. This changed hormonal profile has a responsible role in reduced PR in cattle with low P4 profile. The exact phenomenon responsible for conclusion is still lacking conclusive clarification.

The P4 profile during follicular development was 3.0 ng/mL higher in cows having CL as compared to anovular or cyclic animals with a stage other than diestrus (Bisinotto et al., 2014; Rivera et al., 2011). Elevated P4 profile during the development of ovulatory follicle decreases follicular growth rate (Cerri et al., 2011), improves IGF-1 concentration in follicular fluid as well as embryo quality (Rivera et al., 2011) and subsequent PR (Bisinotto et al., 2013). In lactating dairy cow, this increase in P4 profile was 0.8 ng/mL after single P4 device insertion (Lima et al., 2009). This was considered as low and inadequate compared with the cows having CL (Cerri et al., 2011) to alter the OFD in cows submitted for FTAI (Colazo et al., 2013; El-Zarkouny et al., 2004) or AI at detected estrus (Bisinotto et al., 2015; Lima et al., 2009).

In present study, overall pregnancy loss on d90 post FTAI was lower in OVP5 and OVP7 groups (8 and 13 %) as compared to OVP0 group (17%). In a similar study, Wiltbank et al. (2011) has observed significantly (P<0.05) reduced pregnancy loss (6.8%) in cows having elevated P4 profile in comparison with elevated loss (14.3%) in group with reduced P4 concentration. Stevenson et al. (2006) found no beneficial effect of P4 supplementation on pregnancy loss, but concluded that this loss was minimal in cyclic cows (16%) as compared to non-cyclic cows (31%). In another study, Chebel et al. (2010) compared pregnancy loss in 7 lactating dairy (n=3248) herds. They observed pregnancy loss of 0-17% in Ovsynch protocol as compared to CIDR supplemented cows with 4-12%. Recently, it has been documented that 19.70% pregnancy loss (p>0.05) was observed in dairy cows (n=2207) synchronized with Ovsynch protocol in comparison with Ovsynch+CIDR insert (from 0-7 days of Ovsynch protocol) where pregnancy loss was reported to be 18.7% (El-Tarabany, 2016).

In present study, OFD was non-significantly different (P=0.0711) in all three synchronization groups (OVP0, OVP5 and OVP7). The PR was, although, 7.4% higher in OVP5 group than OVP0 group. Percentage PR was comparable in OVP7 and OVP0 groups. The reason behind this similar PR is might be due to the same physiological status, cyclic, of enrolled animals in all treatment groups. In a previous study, Souza et al. (2007) observed that higher PR (52.6%) was achieved on d60 post AI in animals’ group with medium sized follicles, 15-19 mm, in comparison with smaller, ≤13 mm, and larger sized, ≥20 mm, follicle groups having a PR of 38.2 and 34.4%, respectively. In this study, they used E2 (estradiol-17β) 8h prior to second GnRH.

In present study, progesterone concentration was investigated on d30 and d60 post FTAI. On d30 and d60 post AI, lower P4 values were measured in the cows of OVP0 group as compared to OVP5 and OVP7 groups, but these differences at d30 post FTAI were statistically significant (P<0.05) while on d60 post FTAI, these were found to be non-significant (P>0.05). In a similar research Chebel et al. (2010) found that inclusion of P4 in FTAI protocol has been resulted in enhanced ovulation synchrony. On the other hand, no beneficial impact of CIDR supplementation was observed in comparison with controll group (Lima et al., 2009). In the same study, they found that when Ovsynch protocol was supplemented with CIDR, it was encountered by less chances of premature luteal tissue regression. As reduced P4 profile prior to ovulation and insemination has resulted in premature upregulation of endometrial receptors for E2 and oxytocin which are key factors for PGF2α synthesis and subsequent CL regression (Inskeep, 2004).

Reduced circulatory P4 profile during the estrous cycle preceding insemination has been shown to result in decreased fertility in dairy cows. In similar fashion, Townson et al. (2002) has shown that dairy cows ovulated in third follicular wave have been resulted in higher PR as compared to two waves (81 vs. 63%, respectively; P=0.058). There was a clear positive linear correlation between circulatory P4 profile on the day of luteolysis and successive embryonic viability (Diskin et al., 2002). Shaham-Abalancy et al. (2001) has shown that the outcome of reduced P4 profile was impediment in stimulatory effect on uterine sensitivity to oxytocin during the late luteal phase of the subsequent cycle. As a consequence increased PGF2α may hinder CL maintenance during early embryonic stages. Optimal P4 profile is compulsory for pregnancy establishment and its maintenance but there is lack of information about the minimum threshold level of P4 and embryonic losses. For normal pregnancy maintenance, no cut-off value of P4 has been established hitherto. Many researchers have clearly demonstrated that reduced circulatory or delayed rise of P4 after ovulation is a key factor responsible for embryonic survivability in cattle. Transport of P4 to the uterus is supposed to occur via a local, countercurrent mechanism, arrangement. This was supported by the fact of elevated P4 profile in ipsilateral horn (Weems et al., 1988). Similarly, Stronge et al. (2005) observed that milk P4 profile from d4-d7 post-ovulation has a positive association with embryonic survival in both dairy cattle and heifers. The present study is different from the above study as progesterone concentration was evaluated after d30 and d60 post FTAI.

Conclusion

Proper reproductive management has a vital role for optimal PR in postpartum dairy cattle worldwide. In order to verify P4 level during follicular development, as developing follicle has a profound impact on future reproduction, CIDR inserts were used for a variable period of time with OVP0 protocol. In conclusion, OVP5 protocol can be used as a postpartum reproductive management tool to increase PR. Results of current study have shown an advantage of P4 supplementation as a part of the Ovsynch protocol in cyclic animals either for 5 or 7 days. In both treatment groups, P4 profile was significantly higher than the control group on d30 post FTAI. To minimize the cost, synchronization protocol must be economical and practical as well. The average price of OVP0 protocol is about 1800 PKR and OVP5 or OVP7 is 3000 PKR. As residual conc. of P4 is 0.74 mg after single use for a 7 day period, reuse of autoclaved or disinfected CIDR supplementation (Muth-Spurlock et al., 2016) can reduce the price at 2400 PKR.

Acknowledgments

Authors are grateful to Higher Education Commission, Islamabad for the award of fellowship under Indigenous 5000 PhD Fellowship Program Batch–VII.

Statement of conflict of interest

Authors have declared no conflict of interest.

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