The Effect of A PRID®Delta-Based Estrus Synchronization Protocol On Pregnancy Rates In Cows With No Estrus Symptoms

Murat Can Demir (  murat_can_demir@hotmail.com ) Faculty of Veterinary, University of Kafkas https://orcid.org/0000-0002-3803-069X Cihan Kaçar Faculty of Veterinary, University of Kafkas Umut Çağın Arı Faculty of University of Kafkas Semra Kaya Faculty of Veterinary, University of Kafkas Oğuz Merhan Faculty of Veterinary, University of Kafkas Mushap Kuru Faculty of Veterinary, University of Kafkas Savaş Yıldız Faculty of University of Kafkas


Introduction
Due to the increase in milk yield in cows in recent years, observing estrus symptoms has been challenging. Accordingly, pregnancy rates at the rst insemination have decreased to 35%. Observing the symptoms of estrus and applying arti cial insemination on time are essential to increasing fertility. Due to the di culties in determining estrus symptoms, timed arti cial insemination protocols have been developed.
Research has shown that many factors affect ovarian functions in cows in the postpartum period. These factors include inadequate food intake in the pre and postpartum periods (Baruselli et al., 2004, Opsomer et al., 2000, a negative energy balance, and low body reserve in connection with high milk yield and feed intake in the early postpartum period (Leroy et al., 2008;Beam and Butler, 1999;Opsomer et al., 2000). The high metabolic clearance rate of steroid hormones (Wiltbank et al., 2006), the lower circulating concentrations of progesterone and estradiol in lactating cows compared to non-lactating cows (Sartori et al., 2004;Sangsritavong et al., 2002), and low luteinizing hormone (LH) secretion due to stress and malnutrition (Butler and Smith, 1989) are also in uential. Ovulation is delayed due to the decrease in estrogen production of the dominant follicle due to the inhibition of LH release frequency and suppression of blood glucose, insulin, and insulin-like growth factor-1 (IGF-1) concentrations in cows (Butler, 2000).
It is ideal to have a minimum negative energy balance in the postpartum period. The duration of postpartum anestrus is determined not by the emergence of follicular waves but by follicular deviation and the dominant follicle. Under optimum conditions, follicular growth is a cycle formed by the selection of a dominant follicle, maturation of the follicle, luteolysis, and resumption of ovarian activity, shaping pregnancy. Failure of any of these processes prolongs postpartum anestrus. Cows that do not exhibit any estrus symptoms or have mild estrus symptoms can have an increased incidence of anestrus (Peter et al., 2009). Different hormone combinations have been used in cows with postpartum anestrus, including gonadotropin-releasing hormone (GnRH) with the Ovsynch protocol (Kacar et al., 2006;Bartolome et al., 2005), and progesterone-estradiol-prostaglandin F2α (PGF2α)-equine chorionic gonadotropin (eCG) The present study aimed to determine the effects of progesterone (PRID ® Delta )-based GnRH, PGF2α, and eCG injections on progesterone pro les and pregnancy rates in cows with no estrus symptoms within 60 days after birth.

Animals
The present study was conducted at the Kafkas University Veterinary Faculty Ali Rıza AKSOY Research and Application Farm in Kars, Turkey, with 80 cows that did not show any estrus symptoms within two months after giving birth. Brown Swiss cows, housed in a closed barn system, fed a mixed ration (meadow grass, sainfoin, cattle milk feed, molasses) and with an average daily milk yield of 13-17 liters during lactation, were included in the study. Body condition scores (BCS) were evaluated using a 5-point grading system according to the de nition given by Edmonson et al., (1989).

Protocol
Cows that did not show any estrus symptoms within two months after parturition were included in the study after their ovaries were examined by ultrasound (Draminski Iscan, Poland 7.5 MHz). The selected cows were randomly divided into two groups. The rst group (n = 40) received 2 ml GnRH injected intramuscularly (Ovarelin®, Ceva, Istanbul, Turkey) on day 0, and the intravaginal progesterone-releasing device, PRID ® Delta (1.55 g progesterone, Ceva, Istanbul, Turkey) was placed using an applicator after the lips of the vulva were cleaned with a cotton ball soaked in Baticonol antiseptic solution. The applicator was also cleaned with Baticonol and water before each application. Lubricating gel was applied, and PRID ® Delta was placed into the vagina. On day 8, one day before the removal of PRID ® Delta, 5 ml of PGF2α (Estrumate® RTM, Ceva, Istanbul, Turkey) was injected intramuscularly. PRID ® Delta was removed from the vagina on the ninth day, and the cows were inseminated rectovaginally at the following 60th hour. The second group of cows (n = 40) received 600 IU of eCG, intramuscularly (Oviser®, Hipra, Istanbul Turkey) upon removal of PRID ® Delta, and arti cial insemination was performed rectovaginally 60 hours later ( Figure 1).
To determine the progesterone levels created by PRID ® Delta, 5-ml blood samples were taken from the tail vein into EDTA-free tubes using vacuum injectors on day 0 (the day PRID ® Delta was applied) and day 8 (just before the PGF2α injection). After disinfection, the procedures were carried out using disposable needles on appropriately restrained animals, which is the most practical and risk-free method for both the animal and practitioner.

Obtaining the Blood Serum
The blood samples were centrifuged at 3000 rpm for 10 minutes, and the obtained sera were stored at -20 0 C until analysis. Serum progesterone levels were measured at Kafkas University Faculty of Veterinary Medicine, Department of Biochemistry using commercial ELISA kits.

Statistical Analysis
Statistical analysis of the data was carried out using SPSS ® software (SPSS 20, IL, USA). Normal distribution was assessed with the Kolmogorov-Smirnov test. Follicle diameters between the groups were compared using the Mann-Whitney U test. Pregnancy rates were compared using a chi-square analysis program. Changes in progesterone levels within the group were compared with the repeated measures ANOVA test. Comparison of progesterone levels between groups was made with the Student's ttest if parametric and with the Mann-Whitney U test if non-parametric. Results were analyzed as X±SE.
Values with P <0.05 were considered statistically signi cant.

Results
In cows with no estrus symptoms, the pregnancy rate was 37.5% in the eCG+ group, while it was 27.5% in the eCG-group. Although there was a 10% difference between the two groups, it was not statistically signi cant (P = 0.4, Table 1). The dominant follicle diameter was 15.5 mm in eCG+ cows injected during timed arti cial insemination and 12.4 mm in eCG-cows. Thus, the dominant follicle diameter in cows treated with eCG was much larger, and a statistical difference was found between the groups in terms of follicle size (P <0.001, Table 1). With the application of the progesterone-releasing intravaginal device (PRID ® Delta), serum progesterone concentrations below 1 ng/ml increased above 1 ng/ml on days 8 and 9. No differences were determined in serum progesterone values in blood samples taken until the time of arti cial insemination. However, the progesterone values in the serum taken during the arti cial insemination application were 0.94 ng/ml in the eCG-group and 0.72 ng/ml in the eCG+ group. It was observed that the progesterone concentration during arti cial insemination was much lower in the group in which the eCG was applied with a statistically signi cant difference (P <0.05) ( Figure 2).
No differences were found in pregnancy rates in cows with BCS ≥ 2.75 or < 2.75 in either group. Similar results were found when eCG+ and eCG-groups were compared (Table 2).  . It has also been reported that Nelore heifers treated with eCG had more ovarian response and pregnancy than heifers not treated with eCG (Sá Filho, Torres-Júnior, et al., 2010) Similarly, in Nelore cows in the early postpartum period undergoing progesterone-based timed arti cial insemination with appropriate body condition, it was observed that eCG application and removal of the calf from the mother did not affect the pregnancy rate (Pinheiro et al., 2009). In addition, eCG treatment in the rst synchronized estrus improved reproductive performance in the short term; however, it did not affect it after subsequent inseminations (Rowe et al., 2019). In another study, no signi cant differences were found between the follicular dynamics of cyclic and anestrus groups treated with TCR (56 hours breastfeeding) or eCG, despite the differences in the cyclicity of the animals. Follicular diameter, follicular growth rate, corpus luteum size, and P4 concentrations of anestrous cows treated with eCG or TCR were similar (Barreiros et al., 2014).
In the present study, the effect of eCG injection on pregnancy rates after intravaginal application with progesterone, GnRH, and PGF2α-based protocols and timed arti cial insemination was evaluated in Brown Swiss cows. With the removal of the progesterone-releasing intravaginal device and application of eCG, development of the dominant follicle was accelerated, and ovulation was stimulated with the LH peak. According to the present study results, pregnancy rates were 10% higher in cows treated with eCG; however, no statistical differences were found. The higher pregnancy rate was associated with possible corpus luteum lysis with PGF2α injection one day before PRID®Delta removal and accelerated follicle development with eCG application. The research was conducted on cows that did not show any It has been reported that eCG application during the removal of the vaginal device in cows with anestrus increased the ovulation rate and improved luteal functions (Núñez-Olivera et al., 2014) and eGG administration can reduce differences in ovulation timing (Cavalieri et al., 1997). However, another study reported that ovulation rates did not increase after eCG (Pulley et al., 2013). The eCG treatment yields a larger corpus luteum and higher progesterone concentrations in cows that ovulate after synchronization (Núñez-Olivera et al., 2014). In the present study, it was seen that intravaginal devices containing progesterone could increase serum progesterone concentration in cows without estrus symptoms. For the cyclic activity to develop healthily and the follicular activity in the ovary to continue, the genital organs must be under the in uence of progesterone for a certain period. The progesterone value was lower in cows treated with eCG during arti cial insemination. The low level of progesterone leads to the peak of LH release and increases the likelihood of ovulation. This low concentration of progesterone may have caused the difference in pregnancy rates.
Long-term progesterone treatment did not increase the pregnancy rate in cows with high BCS; however, the combination of long-term progesterone and eCG treatment increased the pregnancy rate per preovulatory follicle and arti cial insemination in cows with low BCS (Bilbao et al., 2015). The effect of eCG applications on fertility in lactating Holstein cows was limited to cows with low BCS. The eCG treatment improved ovulation rates or corpus luteum development, especially in cows with low VKS, and it has been reported that this may lead to increased fertility in cows. It has been observed that cows with a BCS <2.75 have increased follicle growth and fertility with eCG treatment in those with insu cient LH support ). In the present study, no differences were found in pregnancy rates of anestrus cows with or without eCG injection, with a VKS ≥ 2.75 or a VKS < 2.75. Different factors affect the anestrus mechanism in cows other than VKS. In lactating dairy cows under the Presynch-Ovsynch program, eCG treatment at a dose of 400 IU three days before arti cial insemination at the rst service did not increase the size of the largest follicle, follicle growth rate, or pregnancy rate 48 hours after application. It was thought that the dose and timing of eCG treatment could be delayed to affect the maturation period of the preovulatory follicle (Pulley et al., 2013). One study has reported that eCG application did not increase the ovulation rate. However, in another study, it was found that follicle size, corpus luteum volume, serum P4 concentrations, and pregnancy rates were higher when eCG was applied two days before the removal of the intravaginal progesterone device rather than when eCG was applied during the removal of the device in cows undergoing timed arti cial insemination. The most important result obtained in the present study was that the follicle diameter at the time of arti cial insemination was larger in cows that did not show signs of estrus with eCG injection compared to cows that were not injected with eCG. It was observed that this difference in follicle diameter did not affect pregnancy rates, however, it is thought that eCG application increased the growth rate of the follicle in anestrous cows. The larger diameter of the largest follicle in the eCG group can be explained by the fact that eCG increases FSH and LH release for follicle development. The increase in follicular diameter induced by eCG may be related to the positive effect on steroidogenic cells in the ovulatory follicle and the formation of a more competent corpus luteum observed in the treated animals (Núñez-Olivera et al.,

2014).
In conclusion, it was determined that progesterone (PRID ® Delta)-based GnRH, PGF2α application in addition to eCG injections increased the pregnancy rate numerically in cows without symptoms of estrus.
It was also determined that the largest follicle diameter was greater in cows treated with eCG during arti cial insemination; however, follicle size did not increase the pregnancy rate. Hormone application protocol prior to arti cial insemination Progesterone values with and without eCG (Group I and Group II) treatment in cows without symptoms of estrus