Kisspeptin Modulates Luteinizing Hormone Release and Ovarian Follicular Dynamics in Pre-pubertal and Adult Murrah Buffaloes

Abstract

Kisspeptin is a neuropeptide that governs the reproductive axis upstream to GnRH. We wanted to study whether kisspeptin modulates plasma LH and FSH levels and ovarian follicular dynamics in buffaloes and whether kisspeptin can be used for fixed time artificial insemination (FTAI). We carried out these studies in comparison with buserelin, a potent GnRH agonist. Kisspeptin dose-dependently increased plasma LH levels. However, the kisspeptin-induced increase in LH was short-lived as the peak reached in 15-30 min returned to basal values by 1-2 h. The kisspeptin-induced increase in LH level was less compared to buserelin-induced increase in LH level which sustained over time. Kisspeptin did not enhance FSH release while buserelin resulted in a gradual increase over time. LH response to repeated injections of kisspeptin was greater than that induced by buserelin. While buserelin induced an increase in the number of follicles, kisspeptin induced an increase in the growth rate of the follicle. In adult cycling animals, while both the drugs increased plasma LH levels, the increase was greater in buserelin group compared to kisspeptin group. In contrast to the findings in pre-pubertal animals, kisspeptin induced an increase in both the number as well as the size of follicles compared to buserelin. Our studies on oestrus synchronization, using either kisspeptin-PGF_2α-kisspeptin protocol or buserelin-PGF_2α-buserelin Ovsynch protocol on day 0, 7, and 9, respectively, revealed that kisspeptin increased the number of follicles at wave emergence and the diameter of dominant follicle after 2nd dose of drug, the oestrus response rate and duration of oestrus, compared to buserelin. However, conception rate was not significantly different among the groups. From our studies, it appears that Kp and Buserelin differentially modulate follicular dynamics depending on the reproductive age of the animals.However, studies in a larger herd are required to confirm whether kisspeptin can be used for oestrous synchronization in buffaloes.

Keywords: FSH; LH; Murrah buffaloes; buserelin; follicular dynamics; kisspeptin; oestrous synchronization.

Figure 1

Schematic representation of methodology for dose-response study. Following initial basal observation of follicular dynamics for a week, kisspeptin was administered at three different doses at two routes (i.e., IM or IV) at weekly intervals approximately. A minimum of 4 ultrasound observations of follicular dynamics were carried out on alternate days between two injections, as depicted by the solid black arrow. Buserelin was administered at a single dose rate recommended by the manufacturer via two routes (i.e., IM or IV) at triweekly intervals approximately, as depicted by the longer white arrows.

Figure 2

Schematic representation of Ovsynch protocols tested in Murrah buffaloes for oestrus synchronization using either buserelin or kisspeptin. On day 0 and 9, animals in buserelin group were treated with buserelin (10 μg/animal, IV) while that in kisspeptin group received kisspeptin (20 μg/kg, IV). PGF_2α (500 μg, IV) was administered on day 7. Fixed time AI was done 18–24 h post second dose of buserelin or kisspeptin.

Figure 3

Effect of kisspeptin or buserelin on plasma LH and FSH levels in pre-pubertal buffaloes out of the breeding season. (A) Kisspeptin IM dose-dependently elevated plasma LH levels in pre-pubertal buffaloes. However, the LH level reached basal values by 2 h. Buserelin resulted in a gradual but sustained increase in LH level (*kisspeptin 20 μg/kg: basal vs. at 30 min; kisspeptin: 5 vs. 20 μg/kg at 30 min; ϕbuserelin: basal vs. at 2, 3, and 4 h; buserelin vs. all kisspeptin groups at 2, 3, and 4 h time points; P < 0.05, RM ANOVA followed by Dunnett's multiple comparison test). (B) The release kinetics of LH upon IV injections of the drugs were similar to that of IM injections of drugs. (*buserelin: basal vs. at 15 min; kisspeptin: basal vs. kisspeptin 10 and 20 μg/kg at 15 min; Ψkisspeptin 20 μg/kg: basal vs. at 30 min; ϕbuserelin: basal vs. at 1, 1.5, and 2 h; buserelin vs. kisspeptin 5 and 10 μg/kg at 1 h; buserelin vs. kisspeptin 5, 10, and 20 μg/kg at 1.5 and 2 h; P < 0.05, RM ANOVA followed by Dunnett's multiple comparison test). (C) There was no significant increase in plasma FSH upon IM injections of both drugs. (D) Buserelin significantly elevated plasma FSH at 1.5 and 2 h post-injection, however, it was not significantly different from kisspeptin group (*buserelin: basal vs. at 1.5 and 2 h; P < 0.05, RM ANOVA followed by Dunnett's multiple comparison test). Data represented as mean ± SEM (n = 5–6 animals at each time point).

Figure 4

Effect of repeated administration of kisspeptin on plasma LH and FSH. (A) Repeated administration of kisspeptin (10 μg/kg) elevated LH in a sustained manner (*vs. basal; P < 0.05, one-way RM ANOVA). (B) There was no significant increase in FSH level upon repeated administration of kisspeptin. (C) LH levels upon repeated kisspeptin administration were higher than that upon a single injection of buserelin (*kisspeptin: basal vs. all time points; ϕ buserelin: basal vs. 1.5 and 2 h; Ψ buserelin vs. kisspeptin at 30 min; RM ANOVA followed by Dunnett's and Sidak's multiple comparison test). Data represented as mean ± SEM (n = 3–5 animals at each time point).

Figure 5

Alteration in the plasma LH and FSH levels in adult Murrah buffaloes by buserelin and kisspeptin. Experimental buffaloes that received either kisspeptin (20 μg/kg body weight, IV) or buserelin (10 μg/animal, IV) on 11th or 8th day of estrous cycle (i.e., correlating with emergence of waves depending on whether the oestrous cycle is of two-wave or three-wave pattern, respectively) were sampled before (basal) and after drug administration (post-drug samples were collected at the peak time-point as observed in pre-pubertal animals: at 15 min after kisspeptin and at 2 h after buserelin). (A) While both the drugs increased plasma LH levels (*P < 0.05, t-test), the increase was significantly more in buserelin group (n = 6) compared to kisspeptin group (n = 6) (^#P = 0.02, t-test). (B) Buserelin significantly increased FSH concentration while kisspeptin did not (*, ^#P < 0.05, t-test). Data represented as mean ± SEM.