Effect of mid-luteal administration of non-steroidal aromatase inhibitor, letrozole on serum progesterone concentrations in crossbred cows exhibiting prolonged oestrus

Introduction

Prolonged oestrus is one of the common aberrations of oestrus among crossbred cattle, recording an occurrence of up to 34.16 percent (Arun et al., 2020) [1] . Presence of suprabasal progesterone concentration during oestrus, induced by lactational, nutritional and environmental stress is attributed as a common cause of prolonged oestrus (Singh et al., 2009) [2] . The higher progesterone (P 4 ) level during oestrus reduces LH pulse frequency, due to its inhibitory effect on positive feedback of oestradiol (E 2 ) on the hypothalamus, leading to persistence of pre-ovulatory follicles without ovulation. These animals exhibit secondary signs of oestrus for a longer period, due to continued release of oestradiol-17β (Stock and Fortune, 1993) [3] . Suprabasal progesterone has adverse effect on fertility because of ovulation failure. Due to poor and unfavorable oocyte and embryo development brought on by exposure to a low LH surge and an insufficient luteal profile, conception failure occurs (Bage et al., 2002) [4] . Management of prolonged oestrus has been traditionally attempted using prostaglandins, GnRH and steroid hormones, with variable results. Further, use of steroid hormones has been the subject of controversy in cattle industry; hence the use of non-steroidal drugs becomes the novel area of research. Letrozole, a non-steroidal aromatase inhibitor, inactivates the aromatase enzyme responsible for the synthesis of oestrogens (Cohen et al., 2002) [5] . A luteotropic effect is also attributed to letrozole (Yapura et al., 2013; Aravind et al., 2025) [6,7] , which is due to increased LH secretion and alteration of the proportion of large and small luteal cells in the CL (Abdel et al., 2020; Romerein et al., 2018) [8,9] . This in turn gives higher capacity for the CL to produce progesterone and luteal oxytocin and would respond better to PGF 2 α due to larger number of PG receptors (Boonyaprakob et al., 2003) [10] . Low progesterone concentration attained after luteolysis promotes LH production and early attainment of pre ovulatory follicle size (Bridges and Fortune, 2003) [11] . Thus, letrozole might help to correct the suprabasal P 4 levels noticed during oestrus among prolonged oestrous cows. This in turn would lead to timely ovulation of mature follicle and formation of CL that functions normally. Hence, the present study was undertaken to evaluate how mid-luteal administration of letrozole affects subsequent serum progesterone milieu, which might provide information to develop strategies for management of prolonged oestrus in crossbred cows using letrozole.

Materials and Methods

The study was conducted at University Livestock Farm and Fodder Research Development Scheme (ULF & FRDS), Mannuthy during the period from March 2023 to July 2023. Apparently healthy postpartum crossbred dairy cows of age 3-6 years and parity 1-3, maintained under identical conditions of feeding and management were selected for the study. All the cows with oestrus duration longer than 24 h were considered as having prolonged oestrus (Arun et al., 2020) [1] whereas those with duration 24 h or less were considered as normal.

Design of experiment

All the breedable cows specified in the study were thoroughly monitored for signs of oestrus. Twelve animals with prolonged oestrus formed group I and II of six cows each and another 12 normal oestrous cows were allotted to group III and IV (6 cows each). Blood samples were collected from all the cows on the days of oestrus (Day 0, 1 and 2) for serum P 4 estimation. Blood samples were again collected before treatment on 10 th day of cycle for hormone estimation. Cows were subjected to the following treatment as mentioned in the Table below. As in the previous cycle, blood collection was again done in the ensuing cycle (2 nd cycle) during oestrus and on 10 th day of second cycle.

Blood collection and serum progesterone analysis

A quantity of 5 ml whole blood from each cow was collected by jugular puncture using a sterile18 gauge disposable hypodermic needle. It was transferred to a 15 ml sterile test tube and these test tubes were kept in slanting position for 15 min to allow the whole blood to clot. It was placed in a refrigerator at 4 °C overnight for clot retraction. After overnight storage in refrigerator, samples were centrifuged at 3000 rpm for 10 min and clear supernatant sera were collected into storage vials. The vials were labelled and stored at-20 °C till assay. Serum P 4 concentration in all samples were measured using quantitative ELISA kits (Omega Diagnostics, Scotland, UK), following manufacturer's instructions. The data obtained were tabulated and analysed by One-Way ANOVA using SPSS statistics, version 24.0. software programme.

Results and Discussions

Mean serum P 4 concentration (ng/mL) during different days of oestrus and on 10 th day of cycle among the four groups of cows under study in two consecutive cycles is presented in Table 2. In both the prolonged oestrous groups (I and II), no significant change (p≥0.05) in serum P 4 levels before and after treatment, either on the days of oestrus or during dioestrus (day 10) was noticed. Yapura et al. (2009) [12] also reported no significant difference in plasma P 4 levels on any of the days of cycle between letrozole treated and control animals, after intravenous administration of letrozole on days 5 to 7 of cycle. Yapura et al. (2013) [6] reported that persistent release of letrozole in the body has an impact on its hormonal profiles. Single intravenous administration of letrozole during mid-cycle in the present study might be insufficient to bring about luteotropic effect and consequent changes in hormone profile. Hence, a persistent letrozole releasing system might be required to evoke the expected response in hormonal milieu of prolonged oestrous cows. In both the cycles, mean serum P 4 concentration during oestrus was significantly higher (p≤0.05) in prolonged oestrous groups (I and II) than normal animals (III and IV). In both the cycles, mean serum P 4 concentration on day 10 was significantly lower in prolonged oestrous cows than normal oestrous groups. Similar pattern of difference in serum P 4 concentration between normal and prolonged oestrous cows was reported by Niyas (2017) [13] and (Arun et al., 2020) [1] . Existence of suprabasal P 4 concentration during oestrus (Singh et al., 2009) [2] and luteal deficiency during dioestrus (Chander et al., 2002) [14] was reported earlier. Chander et al. (2002) [14] reported value of 3.12±0.51 ng/mL, as the normal serum P 4 concentration during midluteal phase. Suprabasal P 4 concentration during oestrus could be due to improper luteolysis of CL from the previous cycle, because of decreased PGF 2 α synthesis or from extra-gonadal source like adrenal, when the cows are exposed to stress (Duchens et al., 1995) [15] . Presence of luteal tissue at oestrus might have a significant impact on subsequent luteal phase activity, causing luteal insufficiency during dioestrus (Singh et al., 2012) [16] . Milk yield also contributes to disturbed luteal profile, as a strong unfavourable genetic relationship exists between milk production and fertility (Royal et al., 2000) [17] . Though there existed no significant difference in serum P 4 levels between cycles on the day of oestrus (day 0) in either of the normal oestrous groups (III and IV), there existed significant difference (p≤0.05) on day 10 serum P 4 levels in both the groups between cycles, with higher values during 2 nd cycle. Since the hike is not limited to letrozole treated group alone, such changes cannot be attributed to the effect of the drug. Significant variations in serum P 4 levels between cycles among normal oestrous cows is reported earlier also (Arun, 2019) [18] .

Conclusion

Single intravenous administration of 250 µg letrozole at mid luteal phase was not beneficial to correct the defective serum P 4 milieu among prolonged oestrous cows. Existence of suprabasal P 4 concentration during oestrus and luteal deficiency during dioestrus among prolonged oestrous cows was reinforced in the present study. Letrozole administration at different dose level and route of administration, with suitable vehicle for slow release of the drug might be required to achieve a desired result among prolonged oestrous cows.

Acknowledgement

AcknowledgementThe authors are thankful to Kerala Veterinary and Animal Sciences University, India for providing necessary facilities and fund.

References

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