Effect of administration of human chorionic gonadotropin in ovsynch ovulation synchronisation protocol for the management of repeat breeding syndrome in cows

Introduction

Fertility is the key to a cow's lifetime output, influencing both profitability and overall milk production. Achieving optimal fertility helps shorten calving intervals and reduces the days open. However, uterine infections during the post-partum period can significantly hinder reproductive efficiency. In Indian context, reproductive disorders such as endometritis, anoestrus, and repeat breeding are major factors resulting in reduced dairy productivity. Repeat breeding is characterised by cows failing to conceive despite multiple attempts. Repeat breeder cows have normal or nearly oestrous cycle, yet failed to conceive even after being bred three or more times to a fertile bull or inseminated with fertile semen (Roberts, 1986). The major risk factors of repeat breeding are nutritional imbalances, hormonal aberrations, reproductive tract infections, genetic predispositions and karyotypic aberrations. Subclinical endometritis, untimely AI, delayed ovulation, fertilisation failure and early embryonic mortality are the major identified causes of repeat breeding. Certain causes of repeat breeding such as, untimely AI and delayed ovulation can be managed using hormonal protocols. In field condition, most commonly used hormonal protocols are Ovsynch protocols and its modifications. Ovsynch protocol involves the administration of gonadotropin releasing hormone (GnRH) in the early luteal phase, followed by administration prostaglandin F2α seven days' administration of GnRH and again a second dose of GnRH 48 h after administration of PGF2α. Many studies have used Ovsynch protocols for improving pregnancy rate in repeat breeder cows with pregnancy rate varying from 40 to 60 percent. But there are only few studies using hCG in repeat breeder cows. Human chorionic gonadotropin can act directly on the ovary and is independent of pituitary gland for inducing ovulation. The effect of hCG is longer than endogenous LH and also elevates the progesterone concentration than GnRH treatment (Binversie et al., 2012) [8] . Hence the present study was done to study the effect of administration of hCG in Ovsynch ovulation synchronisation protocol for improving the fertility of the repeat breeder cows.

Materials and Methods

The study was conducted on 34 Repeat Breeder (RB) cows in an organized farm, with a 20% occurrence of repeat breeding over one year. Clinico-gynaecological examination revealed two cows with abnormalities (follicular cyst and cervical abscess), which were excluded from the study. The remaining 32 cows were screened for Subclinical Endometritis (SCE) using the Whiteside test, which identified 7 positive animals. These cows were further tested for cytological endometritis using modified cytobrush technique, with a threshold of ≥5% polymorphonuclear cells (PMN). Twenty-four SCE-free cows were selected and divided into three groups: Group I (Ovsynch protocol), Group II (modified Ovsynch protocol), and Group III (control). Group I cows received 10 μg of buserelin acetate on day 5, 500 μg of cloprostenol sodium on day 12, and a second dose of 10 μg buserelin acetate on day 14. Timed artificial insemination (TAI) was performed 18 hours after the second GnRH dose. Group II cows followed a similar protocol, but on day 14, 1500 IU of hCG was administered instead of the second buserelin dose, with TAI 18 hours later. Group III cows were inseminated on the observed day of oestrus. The onset and duration of oestrus were recorded, and blood samples were collected to evaluate serum oestrogen and progesterone levels. Serum samples were taken on the day of oestrus (for oestrogen), day 10 and 21 post-AI, and on days 5, 12, and 14 of the oestrous cycle (for progesterone). The samples were analyzed for hormone levels using Radioimmunoassay (RIA). Ultrasonographic examinations were conducted using a 5 MHz transrectal probe to study ovarian and uterine structures. Measurements included endometrial thickness on days of oestrus, day 5, day 12, and day 14 of the oestrous cycle, as well as follicle size and corpus luteum (CL) diameter on different days. Pregnancy was diagnosed ultrasonographically on day 30 post-AI and confirmed on day 50 by per-rectal examination.

EC-Endometrial epithelial cells

Results and Discussion Occurrence of subclinical endometritis among repeat breeder cows

The overall occurrence of SCE was 21.87 percent, while the occurrence of cytological endometritis was 9.37 percent. Zarekar (2014) [40] and Parikh (2021) [24] reported a similar occurrence of 20.62 and18.42 percent of SCE in RB cows. Pothmann et al. (2015) [25] reported a 12.70 percent prevalence of cytological endometritis in RB cows, with a threshold of PMN cells ≥ 5 percent. In contrary to this, Gupta et al. (2011) [13] , Raja et al. (2012) [28] and Bhat et al. (2014) [6] diagnosed SCE in RB cows using white side test and found an incidence rate of 45.83, 72 and 71.25 percent, respectively. In this study, the lower occurrence of SCE might be attributed to the reduced frequency of reproductive and metabolic issues in the cattle. Additionally, better hygiene practices, optimal breeding management, or the use of repeated treatments and therapeutic interventions may have contributed to the prevention and control of uterine infections.

Time taken for onset of oestrus in group I and group II

All the RB cows in group I and group II responded to Ovsynch and modified Ovsynch ovulation synchronisation protocols. the time taken for onset of oestrus following PGF2α administration for cows in group I was group II was 59.38 ± 1.21 h, while for group II, it was 57.13 ± 1.06 h. No significant difference was observed in the time taken for the time taken for onset of oestrus between RB cows in group I and II (Table 1). Similarly, Ahmed et al. (2016) [2] and Aruna (2021) [2] reported that Ovsynch treatment in cows resulted in 100 percent induction of oestrus after the PGF2α injection. Selvaraju et al. (2008) [31] noted that the time taken for the onset of oestrus after treatment with PGF2α in RB cows was 59.38 ± 0.81, which was in accordance with the present findings. Similarly, Krishnakumar and Chandrahasan (2012) [19] observed a similar time period of 60.20 ± 1.41 h for onset of oestrus after PGF2α administration in RB cows. But, Prajapati et al. (2015) [26] observed a slightly higher duration of 62.10 ± 2.26 h in RB cows treated with Ovsynch protocol. Whereas, Ahmed et al. (2016) [2] observed that RB cows treated with Ovsynch protocol had a shorter period for induction of oestrus (48.750 ± 0.71 h) in RB cows after PGF2α administration. [20] observed that the duration of oestrus in repeat breeder cattle was 28.90 ± 0.14, which is in accordance with the findings of present study. Similarly, Layek et al. (2013) [22] observed that there was no significant difference (p>0.05) between the duration of spontaneous and induced oestrus. But, Bhat and Bhattacharya (2012) [5] observed a duration of 34.43 ± 3.67 h oestrus in RB cows, which is higher than that observed in present study. In contrary to this, Reddy et al. (2022) [29] observed a shorter duration of 22.94 ± 0.87 h in RB cows. In RB cows the extended duration of oestrus might be due to delayed ovulation, likely linked to suprabasal progesterone levels. This delay in ovulation reduced LH pulse frequency, resulting in prolonged oestrogen production and a longer oestrus period (Bigelow and Fortune, 1998) [7] . The shorter duration of oestrus after ovulation synchronisation protocols might be due to administration of hCG or GnRH after PGF2α, which accelerated ovulation, reduced the oestradiol peak, and shortened behavioural oestrus.

Endometrial thickness on different days of oestrous cycle

There was significant difference (p<0.01) in endometrial thickness on day 0, day 5 and day 12 of oestrous cycle within both group I and group II (Table 3 and Fig. 2 to 5). There was significant difference (p<0.05) in the endometrial thickness on day 5 between group I (5.15 ± 0.25) and II (4.50 ± 0.09). This might be due to the higher progesterone level in animals in group II (8.94 ± 1.33) compared to animals in group I (6.12 ± 1.69). DesCoteaux et al. (2010) noted that ultrasonographic image of uterus during peri-oestrus phase showed noticeable swelling, more heterogenous appearance and less uniform gray tones of the uterine wall, which is in accordance with the present study. Ahmadi et al. (2019) noted that uterus was thicker during the oestrus than dioestrus phase of the oestrous cycle. Increased endometrial thickness on day of oestrus might due to high level of oestrogen in circulation and reduced endometrial thickness during luteal period might be due to high circulating level of progesterone during luteal phase of oestrous cycle. DesCoteaux et al. (2010) observed that the high level of progesterone in circulation decreases the endometrial thickness. ---** Significant at 0.01 level; * Significant at 0.05 level; ns -non-significant Group I -Ovsynch, Group II -modified Ovsynch, Group III -Control One-way ANOVA followed by DMRT, Independent t-test, Repeated Measures ANOVA followed by LSD

Number and diameter of follicles on different days of oestrous cycle in RB cows

The number of small follicles in group III on day 0 was significantly higher (p<0.01) than group I and II. There was significant difference (p<0.05) in the diameter of medium sized follicle in group II with group I. There was no significant difference in the number and diameter of medium and large sized follicles on day 0 between groups. On day 5 of and day 12 oestrous cycle, there was no significant difference (p>0.05) in the number of different sized follicles and diameter of small and large sized follicles, between group I and group II. The size of the dominant follicle during induced oestrus was 12.06 ± 0.38 mm for group I and 12.16 ± 0.39 for group II. Diameter of dominant follicle on day of spontaneous oestrus was 10.96 ± 0.31 for group III and there was no significant difference (p>0.05) in the diameter of dominant follicle during induced oestrus in group I and II (Fig. 6 to 7). Bage et al. (2002) [4] found that RB cows had more small follicles in their ovulatory wave compared to virgin heifers. Lamb et al. (2003) [21] reported significantly higher pregnancy rates (p<0.01) in cows with follicles larger than 12 mm. Similarly, Keskin et al. (2010) [18] noted that cows with ovulatory follicles between 13.50 and 16.50 mm had higher conception rates and lower embryonic loss. Singh et al. (2019) [34] observed larger dominant follicles was observed in Ovsynch treated RB cows compared to controls, aligning with findings of current study. Kashyap et al. (2020) [16] observed that in repeat breeder cows treated with Ovsynch protocol the average diameter of preovulatory follicle measured 11.48 ± 0.44 mm, supporting the findings of the present study.

Diameter of CL oestrous cycle on different days of oestrous cycle in RB cows

The diameter of CL on day 5 for group I and II was 9.96 ± 2.19 and 14.10 ± 0.43 mm, respectively and on day 12 it was 18.51 ± 0.73 and 19.31 ± 0.46 mm, respectively. There was no significant difference (p>0.05) in the diameter of CL between group I and II on day 5 and day 12. Diameter of CL day 10 post AI in group I (19.28 ± 0.49) and II (19.76 ± 0.46) was significantly higher (p<0.01) than group III (17.79 ± 0.47). There was no significant difference (p>0.05) in the diameter of CL on day 21 post AI between post AI (Table 6 and Fig. 8 to 9). The larger diameter of CL in the cows treated with GnRH, along with the higher circulating progesterone levels compared to untreated animals could be due to an enhanced CL function, which might positively affect fertility (Hasan et al., 2021) [14] . Treatment with hCG in cows has been linked to an increased number of large luteal cells and a reduction in small luteal cells, contributing to a larger CL (De Rensis et al., 2008) [12] . The greater CL diameter day 10 post AI in group I and II compared to the group III, might be due to the luteotropic effect of GnRH or hCG administered in the treatment groups. --** Significant at 0.01 level; ns -non significant Group I -Ovsynch, Group II -modified Ovsynch, Group III -Control One-way ANOVA followed by DMRT, Independent t-test

Serum progesterone concentration on different days of oestrous cycle

The serum progesterone concentrations on the day of oestrus were 1.71 ± 0.44, 2.49 ± 0.22, and 2.21 ± 0.24 for groups I, II, and III, respectively. Suprabasal progesterone concentrations (>1 ng/mL) were observed in all three groups, with no significant difference across the groups (p>0.05). Bage et al. (2002) [4] observed similar suprabasal progesterone levels in repeat breeders, and Singh et al. (2005) [32] also noted elevated levels in repeat breeder heifers. Suprabasal progesterone might result from incomplete regression of the Corpus Luteum (CL) and low prostaglandin levels, negatively affecting ovulation and LH surge, thus impacting fertility (Bigelow and Fortune, 1998) [7] . Additionally, higher progesterone levels might be produced by the adrenal glands during stress (Honparkhe et al., 2010) [15] . On day 5 of the oestrous cycle, progesterone concentrations were 6.12 ± 1.69 and 8.94 ± 1.33 for groups I and II, respectively. The progesterone concentration on day 10 post AI was significantly higher in group II (12.83 ± 1.32) compared to groups I (8.22 ± 1.07) and III (7.97 ± 1.13), with p<0.05. Progesterone levels further increased on day 12 (10.55 ± 2.99 and 15.07 ± 2.55 for groups I and II) and decreased by day 14 (1.40 ± 0.40 and 1.29 ± 0.45 for groups I and II). The findings were consistent with Murugavel et al. (2018), who reported lower progesterone levels on day 5 and higher concentrations on day 12 due to the functional CL or luteotropic effects of GnRH. On days 10 and 21 post AI, group II had significantly higher progesterone concentrations (p<0.05) compared to the other groups. These findings align with studies by De Rensis and Peters (1999) [10] and Binversie et al. (2012) [8] , who observed higher progesterone levels in cows treated with hCG compared to GnRH

Serum oestrogen concentration

The mean serum oestrogen concentration was 11.76 ± 1.65, 12.46 ± 1.84 and 13.31 ± 1.45 pg/mL for group I, II and III, respectively. On day of oestrus, there was no significant difference (p>0.05) in the level of oestrogen on the day of oestrus between groups. Sub basal oestrogen concentration (< 10 pg/mL) was observed in 37.50 percent of all RB cows. This finding is in accordance with observations of Sultana et al. (2022) [37] , they found that the mean serum oestrogen level in RB cows as 15.49 ± 2.45 pg/mL. They also noted that the serum oestrogen in normal cycling cows (25.40 ± 4.07 pg/mL) was higher RB cows. But, Sood et al. (2015) [35] observed that RB cows had higher average plasma oestradiol levels (105.20 pg/mL) than normal cycling group, which had a lower average concentration of 85.60 pg/mL. The lower average concentration of oestrogen in RB cows when compared to the normal cycling cows might be due to poor nutrition in farm or metabolic disorders that could affect the endocrine system, resulting in poor follicular development and reduced oestrogen production. Increased steroid metabolism in liver due to lactational stress might be also a reason for the lower oestrogen concentration in RB cows (Wiltbank et al., 2006) [39] .

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