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Synchronization and Resynchronization as a Novel Approach for Improving Reproductive Performance of Postpartum Dairy Cows.

Byline: A. H. Shahzad, A. Sattar, A. Husnain, I. Ahmad, N. Ahmad, D. Nak and Y. Nak

Key words

Postpartum cows, CIDR-EB based synchronization, Progesterone profile, Reproductive management, Pregnancy rate.


Modern dairy industry is facing multiple issues like estrus expression and decreased PR around the globe. The success of commercial dairy farming is based on the optimal inter calving interval (ICI). Hence, cows must return to ovarian cyclicity, express estrus and be bred within 85 days postpartum to achieve ICI of 365 days. Calving is a traumatic event and the ability to control ovarian and uterine events in the postpartum cow could play an important role in achieving subsequent fertility (Mobashar et al., 2018). Two major physiological factors which influence the reproductive success in postpartum dairy cow are: ovarian cyclicity and uterine health. For this purpose, estrus synchronization is a useful reproductive management tool. In recent years; resynchronization in a suitable manner is also a good management tool for improved conception rate (CR).

Early pregnancy diagnosis (EPD) and rebreeding of open cows are crucial to improve reproductive performance and enhanced CR in dairy herds (Fricke et al., 2003). Without early pregnancy diagnosis and resynchronization protocols, there may be extended inter estrus interval, prolonged days in milk (DIM), and decreased milk production (Dewey et al., 2010). In high producing cows, CR is low (< 40%) leading to majority of non-pregnant cows (Fricke et al., 1998). Despite the better understanding of reproductive physiology and lower number of services per conception following by first AI, early postpartum successful breeding is still a goal to be achieved (Galvao et al., 2007; Chebel et al., 2006). This situation becomes worse if the pregnancy diagnosis and resynchronization strategy is not executed in a timely manner especially for the remaining 60% of non-pregnant animals.

After early pregnancy diagnosis, open cows require to be enrolled promptly for the subsequent breeding. To minimize the inter-insemination intervals, an ideal resynchronization protocol needs to be standardized.

A routinely employed resynchronization protocol includes a shot of GnRH to the all cows irrespective of pregnancy status seven days prior to pregnancy evaluation. On pregnancy diagnosis, open cows are subjected to PGF2[alpha] and GnRH at 2 days interval of PGF2[alpha] followed by TAI (Fricke et al., 2003). It is a cost effective method and potential reproductive managemental tool to decrease the interbreeding intervals (Galvao et al., 2013; Giordano et al., 2013; Tenhagen et al., 2004). Reduced pregnancy rates have been reported following resynchronization protocols in comparison with synchronized based TAI. One major reason for reduced PR is improper intervention in estrous cycle with synchronization and resynchronization protocols (Bilby et al., 2013; Bruno et al., 2013). Research data have shown that in resynchronization group, large proportions of cows were not on anticipated stage of the estrous cycle at the time of initial GnRH of Ovsynch (Bisinotto et al., 2010; Bartolome et al., 2009).

Other important explanation for decreased PR following resynchronization protocol is that 15-26% animals don't have functional luteal tissue leading to suboptimal P4 in circulation (Silva et al., 2009; Sterry et al., 2006; Fricke et al., 2003). To overcome lower conception rate, many researchers used CIDR inserts in resynchronization protocol and obtained beneficial impact on PR in lactating dairy cows (Bilby et al., 2013; Bisinotto et al., 2010; Chebel et al., 2010; Dewey et al., 2010).

There is no literature available on the implementation of a CIDR based GnRH resynchronization treatment followed by CIDR-EB synchronization protocol in lactating dairy cows. Therefore, it was hypothesized that a similar treatment used in conjunction with this protocol (CIDR-GnRH-PG-GnRH) followed by TAI will result in a highly synchronized follicular wave emergence and subsequent ovulation in open cows for resynch and increased TAI pregnancy rates in lactating dairy cows with beneficial effects of P4 supplementation on existing pregnancy.

Primary objectives of the present study were to examine the effect of CIDR-EB protocol on PR in lactating dairy cows, 2) to determine the effect of CIDR on PR and P4 supplementation on pregnancy loss and to investigate the CIDR insertion on resynch protocol for insemination of open lactating cows on d33 after US without waiting period for resynchronization, and 3) to compare P4 concentration before and after CIDR insertion and luteal tissue cross-sectional area in control and resynch group.


Experiment I, Location A

Animals, nutrition, and location

Normally calved multiparous lactating Holstein cows (n = 160) with 45-110 DIM, housed in cross-ventilated free stall barns on a commercial dairy farm in Yenisehir Bursa, Turkey (40Adeg15'52"N 29Adeg39'11"E) were used in the present study. Before the start of the study, all the cows were subjected to BCS as described by Ferguson et al. (1994) and cyclicity evaluation through ultrasonography. Cows with BCS ranging 2.50-3.25 were included in the study. Estrus response was evaluated for CIDR-EB protocol. Animals were kept open barns with shade and a yard with a concrete floor under stall barn housing system, fed TMR (total mixed ration consisting of alfalfa hay, straw, maize silage and concentrates) twice daily ad libitum and water. Mineral mixture and salt blocks were made available in the barns. Feed formulation was in accordance with NRC requirements for lactating dairy cattle.

Feed increment was offered to high producing cows accordingly. During the whole course of experiment, cows were milked thrice daily. The experiment was conducted during the months of April to October.

Experimental design

All animals (n = 160) were synchronized using standard CIDR-EB protocol as described previously (Naseer et al., 2011). Cows were inseminated using frozen thawed semen at 48-60 h after CIDR removal. On day 14 post AI, all the cows were randomly allocated to two groups: 1) resynch (n = 90) received previously used autoclaved (Zuluaga et al., 2008) CIDR for 7 days on day 14th post TAI. GnRH was injected to all cows on d23. On d30, all cows were subjected to PD through US. Cows found non-pregnant received 2 ml (im) PGF2[alpha] on the same day and 100 ug (im) GnRH two days later. TAI was done 64-68 h post PG injection 2) control (n = 70) group was only inseminated at detected estrus and no further treatment was carried out (Fig. 1).

Blood sampling and RIA for P4 profile

Blood sampling from all the cows was done on d14, d16 and d30 post TAI. Five ml blood was collected from the coccygeal vein in Vacutainer coated with EDTA (Becton, Dickinson and Co., Franklin Lakes, NJ). Blood samples were spun in a centrifuge @ 2800X g for 20 min to separate plasma. After harvesting the plasma, it was transferred to eppendorf tubes and stored at -20 AdegC till P4 analysis. A subset of plasma samples (resynch; n = 30 and control; n = 27) of the cows found pregnant on day 30 post insemination were further processed for P4 analysis through radio immune assay (RIA) while the remaining stored plasma samples were discarded. Progesterone values were measured using a solid-phase, RIA kit (Coat-a-Count Progesterone, Diagnostic Products Corporation, Los Angeles, CA) described by Patterson et al. (1995).

Pregnancy diagnosis and measurement of luteal tissue cross-sectional area

Pregnancy was diagnosed by ultrasonography at d30, d60 and d90 post TAI. It was confirmed by presence of amniotic vesicle (AV), heartbeat of embryo and intraluminal uterine fluid as pregnancy markers as described by Fricke (2003). Pregnancy losses (%) were recorded on d60 and 90 post TAI. Luteal tissue cross-sectional area was measured in cows which were positive for pregnancy on d30 post TAI. Cross-sectional area of luteal tissue was measured with following formulae (Kastelic et al., 1990):

Area without a cavity = wClxhClxI/4

Area with cavity = (wClx hClx I/4) - (wCx hCx I/4)

Where, w is width of luteal tissue, h is height of luteal tissue and C is cavity inside luteal tissue.

Statistical analyses

Effect of treatments in resynch and control groups on first-service conception rates and overall pregnancy rates were determined by chi square analysis in PROC FREQ of SAS (Statistical Analysis System, Version 9.1 for Windows; SAS Institute, Cary, NC, USA). Unadjusted odd ratios were calculated using the logistic procedure of SAS. Interactions of BCS, parity, cyclicity and estrus response were calculated on pregnancy odds. Effect of treatments on circulatory P4 profile and luteal tissue cross sectional area were analyzed by using GLM procedures of SAS. Differences were declared significant if P < 0.05 and a trend towards significant was supposed when 0.05 a$? P a$? 0.10.

Experiment II, Location B

Animals, nutrition, and location

Normally calved multiparous lactating Holstein cows (n = 118) with 45-110 DIM, housed in cross-ventilated free stall barns on a commercial dairy farm in Chunian Distt. Kasur, Pakistan (30Adeg 58' 0N 73Adeg 58' 60 E) were used in the present study. Multiparous cows with 45-110 DIM were enrolled in this study. Before the start of study, all the cows were subjected to BCS as described by Ferguson et al. (1994) and cyclicity evaluation through ultrasonography. Cows with BCS ranging 2.50-3.25 were included in the study. Animals were kept open barns with shade and a yard with a concrete floor under stall barn housing system, fed TMR (total mixed ration consisting of alfalfa, straw, maize silage and concentrates) twice daily ad libitum and water.

Mineral mixture and salt blocks were made available in barns. Feed formulation was in accordance with NRC requirements for dairy cattle. Food increment was offered to high producers accordingly. The cows were milked twice daily during the experiment which continued from October to July.

Experimental design

Treatments of all the animals (n = 118) in this experiment were same as in study 1 but without blood sampling and luteal tissue measurement. Parameters under study in resynch group (n = 54) were compared with those of control (n = 64) group (standard CIDR-EB protocol; Fig. 2).

Pregnancy diagnosis

Pregnancy was diagnosed by ultrasonography (Aloka 900 V equipped with a 5.0 MHz linear array transducer, Aloka, Wallingford, CT, USA) at d30, d60 and d90 post TAI. It was confirmed by presence of amniotic vesicle (AV), heartbeat of embryo and intraluminal uterine fluid as pregnancy markers as described by Fricke et al. (2003). Pregnancy losses (%) were recorded on d60 and d90 post TAI.

Statistical analyses

Effect of CIDR based synchronization and resynch treatment on first-service conception rates and overall pregnancy rates were determined by chi square analysis in PROC FREQ of SAS (Statistical Analysis System, Version 9.1 for Windows; SAS Institute, Cary, NC, USA). Odd ratios, unadjusted, were calculated using the logistic procedure of SAS. Interactions of BCS, parity, cyclicity and estrus response were calculated on pregnancy odds. Differences were declared significant if P 0.05) difference of progesterone concentration. However, after 48 h, cows of resynch group had significantly (P< 0.05) elevated P4 values as compared to those of control. Similarly, on d30 post AI, P4 values of both groups differed significantly (P< 0.05, Table II).

Table II.- Plasma progesterone concentrations (mean +- SD) on days 14, 16, and 30 for pregnant dairy cows in experiment I.

Days post AI###Treatment1

###Resynch (n = 30)###Control (n = 27)

###14###5.50 +- 1.12###5.38 +- 0.96

###16###6.47* +- 0.99###5.60 +- 1.15

###30###7.57* +- 1.14###6.34 +- 1.32

Luteal tissue cross-sectional area

Based on the mean luteal tissue cross-sectional area, the corpora lutea of the cows in resynch (n = 30) and control (n = 27) group, on d30 post TAI was 422 +- 98 mm2 and 490 +- 127 mm2, respectively (P = 0.03) as shown in Table III.

Table III.- Luteal tissue cross-sectional area (mean +- SD, mm2) for pregnant cows on day 30 post insemination in experiment I.

Resynch (n = 30)###Control (n = 27)

###422* +- 98###490 +- 127

Experiment II

Pregnancy rate and pregnancy loss

The PR, in second study, was observed to be 54% on day 30 post AI in the cows of resynch group which was significantly (P = 0.028) higher than the control group (44%). Similarly, overall PR at d60 and d90 post AI in resynch group of cows was higher (72 and 70%) as compared to those of control group (53 and 50%) and the differences were significantly higher (P = 0.033 and 0.025) in both groups, respectively (Table I). No pregnancy loss was observed between d30 and d60 in resynch group while it was 7% in control group (P = 0.090) of cows. Overall pregnancy loss between d30 and d90 was also reported to be lesser (3%) for cows in resynch group in comparison to control group (14%) with no CIDR insertion post TAI. These pregnancy losses were statistically significant (P= 0.071; Table I) in resynch versus control groups.

Combined pregnancy rate and loss in both studies with different geographical locations

Combined PR in both experiments (I and II) was observed to be 50% on day 30 post AI in the cows of resynch group which was higher (P = 0.26) than the cows of control group (43%). Similarly, overall PR on d60 and d90 post TAI in resynch group of cows (73 and 72%) was higher as compared to those of control group (48.5 and 46%) and the differences were significantly higher (P = 0.00003 and 0.00002) in both groups, respectively (Table I). Between day 30 and 60, pregnancy loss in resynch group was lower as compared to pregnancy loss of 10% in control group (P = 0.12). Overall pregnancy losses between d30 and d90 was reported to be significantly less (P = 0.027; 4%) for cows in resynch group in comparison to control group (13%) with no CIDR insertion post AI (Table I).


In this experiment, two studies were conducted to evaluate whether quick resynchronization protocol would result in an early scheduled synchronized estrus to facilitate the FTAI followed by synchronization in lactating dairy cows deemed open on PD. It was aimed to reduce the optimal time period in resynch groups in both studies. The potential benefit from the anticipated increase in overall PR following resynch protocol response would be an advantage if resulted in increased PR resulting from FTAI. There is no information available about CIDR-EB based and resynch protocols in lactating dairy cows. Major hindrance in using synchronization protocols containing EB (estradiol benzoate) are availability and restrictions on this product in dairy industry. Availability and usage issues make GnRH and gonadotropins (LH, hCG) as products of choice for ovulation induction in synchronization protocols. But in many areas where it is being used has resulted in promising outcomes.

In present study, standard CIDR-EB protocol has been investigated as postpartum reproductive management tool in lactating dairy cows followed by resynch manipulation for open animals on d30 post TAI. In a previous study, Souza et al. (2009) has shown CIDR-EB based synchronization protocol has resulted in emergence of synchronized follicular wave in 84.4% animals within 1-5 days post treatment. Another advantage of using EB in CIDR based protocol, it does not require to check/monitor follicular wave status (Bo et al., 1994) which makes it a protocol of choice in tropical and subtropical regions where heat stress has dominant impact on follicular development resulting in decreased probability of ovulation followed by GnRH based synchronization protocol (Vasconcelos et al., 1999).

Using CIDR in Ovsynch protocol has resulted in elevated P4 concentration leading to lowered LH pulsatility which in turn improves competency of ovulated oocyte (Revah and Butler, 2001) and subsequent improvement in PR (Vasconcelos et al., 1999).

Main objective of the present study on both geographical locations was to evaluate the effect of CIDR insert on post TAI in CIDR-EB based standard protocol and early resynch protocol after the diagnosis of non-pregnant cows in resynch group. In present study, selection of animals was on random basis as the start protocol consisted of CIDR insert. In previous studies, improved pregnancy per AI has been reported (Bilby et al., 2013) in 7-day resynch protocol when either absence of CL (27.0 versus 19.2%) or low (<1 ng/mL) P4 (29.4 versus 15.0%) profile. In present study, pregnancy outcome on location A was 43% and 48% in control and resynch group (P = 0.50), respectively. In our study, resynch protocol was initiated on day 14 post AI by CIDR insert while in another study, Pulley and Stevenson (2015) has shown negative impact of P4 supplementation, when introduced at day 35 post AI, on P/AI.

In another 5-d resynch protocol, Bisinotto et al. (2010) observed tendencies (P = 0.07 to 0.10) for increased P/AI on day 32 and 60 post AI, when resynch protocol was introduced at day 34 post AI, in open cows with CL+CIDR device insert and CL bearing cows without CIDR insert. In another study conducted by Mehni et al. (2012), it was found that insertion of P4 implant in Holstein cows from d5-d19 post AI resulted in increased PR as compared to control group (56 versus 25%). This significant increase in PR was the possible effect of reduced embryonic mortality by elevated P4 profile in treated cows. Similar observations were recorded by Villarroel et al. (2004) who demonstrated a trend towards lower pregnancy loss (P = 0.077) in repeat breeder cows supplemented with P4 implants from d5-d19 post AI in treatment group versus control. In our study, no comparison was possible as we started it on 14 days post TAI when virtually all cows were supposed to have CL.

In present study PR followed by first service at both locations in control groups was less than P4 supplementation groups. A meta-analysis study by Mann and Lamming (1999) has shown that P4 supplementation within one week of AI yielded higher fertility but beyond this in 2-3rd week post AI, P4 failed to have any impact on PR.

Treatment of cows with CIDR inserts during resynchronization protocols improves P/AI to re-insemination by approximately 5 percentage units when the resynchronization protocols were initiated at approximately 38 +- 3 d after AI (Bilby et al., 2013; Dewey et al., 2010). It is not clear, however, whether the use of a CIDR insert during a resynchronization protocol would increase P/AI to re-insemination of cows that had their estrous cycle presynchronized with GnRH or PGF2[alpha]. In present study, overall PR was higher in resynch group on both locations. This is first study of its kind in which CIDR-EB was used for synchronization and CIDR-Ovsynch was used for resynch protocol. Resynchronization with P4 supplementation improves and synchronizes induction rate in non-pregnant cows (Colazo et al., 2006; Stevenson et al., 2003).

This P4 supplementation after 5d post AI improved fertility in Holstein cows (Villarroel et al., 2004), but resulted in decreased fertility when introduced within 48 h of AI (van Cleeff et al., 1996). It is obvious that P4 supplementation could be used to resynchronize cow but introduction timings may influence the survival of earlier established pregnancy and subsequent fertility of the following ovulation of the succeeding estrous.

In present study pregnancy loss at location A was found to be low, between first and second pregnancy check, in resynch group (5.0%; 2/43) compared to control group (13.0%; 4/30). Similarly, overall pregnancy loss between day 30 and 90 was also reduced (P< 0.05) for cows in resynch group (4.0%; 3/68) in comparison to control group (11%; 4/36). On location B pregnancy loss was 0.0% between d30 and d60 in resynch group as compared to control group (7.0%; 2/28) with no CIDR insertion post TAI. Overall pregnancy loss between day 30 and 90 was also reduced (p 0.05). On d16 and d30 P4 profile was significantly elevated in resynch group. Reason on d16 is obvious but on d30 is fortuitously. At the same time luteal tissue area was significantly smaller in resynch group in comparison with control group. This, unexpected, outcome may be due to some synergistic effect of supplemented P4 on luteal progesterone production or GnRH based LH surge dependent supplementary P4 production by small luteal cells (Niswender and Nett, 1988). In literature no data are available for the comparison.

At both locations there was huge difference between two herds on milk production basis. Despite this difference, reproductive performance did not differ in control versus control and resynch protocol at locations A and B. In literature erratic findings have been reported on account of production impact on reproduction. In one study decreased, although slight, reproductive efficiency was documented in high producing group (Lucy, 2001). In current similar weather conditions prevailed at both location. In another review Santos et al. (2004) observed that early and late fetal mortality has no link with high production in dairy cows. Lucy (2001) has shown negative correlation between high yielding and fertility but this might be the result of other contributory factors like increased incidence of postpartum issues and heat stress. In our experiment production factor did not altered fertility which was in accordance to the previous results (Lopez-Gatius, 2003).


In conclusion, standard CIDR-EB based synchronization protocol is practicable synchronization protocol for postpartum dairy cows which has resulted in similar PR at different geographical locations. Resynch intervention is, at the same time, also a viable protocol for early breeding in open cows after pregnancy diagnosis to reduce the days open in lactating dairy cows. Further investigations are warranted at large scale and use of autoclaved CIDR in resynch protocol to make it more economical one. Results of present study also support the hypothesis that P4 supplementation when used, between days 14 and 21 after TAI program, synchronized the subsequent estrus in the majority of non-pregnant lactating cows and reduced embryonic losses for resynch group containing CIDR insert compared to control group.


This project was supported by TUBITAK in experiment I and Higher Education Commission (HEC) Islamabad in experiment II under Indigenous 5000 PhD Fellowship Program Batch-VII. The authors gratefully acknowledge Akbaslaar dairy Yenisehir, Bursa, Turkey and Living Dairy Chunian, Kasur for providing all generous support related to animals' availability and physical support as well.

Statement of conflict of interest

Authors declare that they have no conflict of interest.


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Author:Shahzad, A.H.; Sattar, A.; Husnain, A.; Ahmad, I.; Ahmad, N.; Nak, D.; Nak, Y.
Publication:Pakistan Journal of Zoology
Article Type:Report
Date:Apr 30, 2019
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