Thesis_Pitcher_Desley_J_Redacted.pdf (3.86 MB)
Download file

Pituitary and ovarian function in female cattle treated with Deslorelin, an agonist of Gonadotrophin-releasing hormone

Download (3.86 MB)
thesis
posted on 16.02.2021, 01:00 by Desley J Pitcher
Female cattle (heifers) treated with deslorelin, an agonist of gonadotrophin-releasing hormone(GnRH), typically show a biphasic gonadotrophin response to treatment. During the acute phase (0 to 24 h), plasma concentrations of luteinising hormone (LH) are elevated. The acute phase is followed by GnRH receptor downregulation in the anterior pituitary gland, and the pituitary becomes desensitised to natural sequence GnRH. Heifers treated chronically with deslorelin therefore do not have pulsatile secretion of LH. However, heifers treated chronically with deslorelin commencing early in the oestrous cycle (Day 3), typically had elevated plasma concentrations of progesterone during the luteal phase compared with contemporary untreated heifers. It is unlikely that increased plasma progesterone in heifers treated with deslorelin, commencing early in the oestrous cycle, is due to ovulation in response to the acute rise in plasma LH, and subsequent formation of an accessory corpus luteum. The increase in plasma progesterone in these treated heifers might be due, therefore, to changes in patterns of LH secretion, which are accompanied by changes in the size and/or function of the corpus luteum. Heifers treated with deslorelin commencing later in the oestrous cycle may be induced to ovulate and develop an accessory corpus luteum. The aim in Experiments 1 and 2 of this thesis was to characterise LH secretion, and determine changes in size and steroidogenic activity of the corpus luteum, in heifers treated chronically with deslorelin, commencing on Day 3 of the oestrous cycle. Steroidogenic activity was ascertained by measuring tissue contents of progesterone, steroidogenic acute regulatory (StAR) protein and the key steroidogenic enzymes, cytochrome P450scc (P450scc) and 3ß-hydroxysteroid dehydrogenase, ⁵-⁴isomerase (3ß-HSD). The recently described StAR protein facilitates the transport of cholesterol to the inner mitochondrial membrane of steroidogenic cells, where P450scc is localised. Transport of cholesterol to the inner mitochondrial membrane is considered the ratelimiting step in steroidogenesis in all steroidogenic tissues. A further aim in the thesis was to determine the relationship between stage of the oestrous cycle (i.e. stage of the ovarian follicular wave) and the ability of deslorelin to induce ovulation during the acilte LH response phase. In Experiment 1, stage of the oestrous cycle was synchronised in randomly cycling Brahman heifers, using a standard progest-agen treatment. On Day 3 of the ensuing oestrous cycle, heifers were assigned to: Control (n=9), no treatment; Deslorelin (n=9), implanted subcutaneously with GnRH agonist (deslorelin) bioimplants. Heifers treated with deslorelin received approximately 25 g deslorelin/kg live weight/24 h). Blood samples for LH and progesterone analyses were taken immediately before implanting (Day 0) and 6 hours later, and on Days 1,3, 5, 8, 9, 10. On Day 10 of treatment (Day 13 of the oestrous cycle), all heifers were ovariectomised and the corpus luteum dissected from the ovary and weighed. Immediately after ovariectomy, all heifers received an injection of natural sequence GnRH (50 g i.m.) and blood samples were taken at 0 and 30 min to determine pituitary LH release. Progesterone content of the corpus luteum was determined by radioimmunoassay. Contents of StAR protein, P450scc, and 3ß-HSD were determined using Western Blot analyses. Experiment 2 was similar in design to Experiment 1, except that heifers treated with deslorelin received approximately 50 g deslorelin/kg live weight/24 h. Also, in Experiment 2, heifers were bled on Days 1,3,5,7,9,10. The trends in the treatment responses were similar for Experiments 1 and 2 and pooled data are presented below. Treatment with deslorelin caused an acute increase in the plasma concentration of LH and, at 6h after implanting, treated heifers had greater (P<0.01) plasma LH (1.4 ±0.1 ng/ml) than control heifers (0.6 ± 0.1 ng/ml). Plasma LH in implanted heifers had declined by Day 1; however, mean LH from Day 1 to 10 of treatment was greater (P<0.01) for implanted heifers (0.8 ± 0.1 ng/ml) than control heifers (0.5 ± 0.1 ng/ml). On Day 10, control heifers had an increase (P<0.01) in plasma LH after injection of GnRH (LH 1.73 ± 0.23 ng/ml), but this did not occur in heifers implanted with deslorelin (LH 0.11 ±0.08 ng/ml). Plasma progesterone concentrations increased from Day 0 to Day 10 for both control heifers and heifers treated with deslorelin. On Day 10, plasma progesterone was greater (P<0.01) for heifers implanted with deslorelin (18.9 ± 3.5 ng/ml) than control heifers (9.1 ± 1.3 ng/ml). Corpus luteum weight was greater (P<0.05) for implanted (4.2 ±0.4 g) than control (3.1 ± 0.2 g) heifers. The amount of progesterone per corpus luteum was also greater (P0.01l) for treated heifers (164 ±20 g per corpus luteum) than control heifers (88 ± 13 g per corpus luteum). The amount of StAR protein per total corpus luteum was greater (P<0.05) for heifers treated with deslorelin (2.2 ±0.3 arbitrary units) than control heifers (1.2 ± 0.2 arbitrary units). The relative content of StAR protein per unit weight of corpus luteum was greater in treated heifers (1.8 ± 0.3 arbitrary units/g) compared with control heifers (1.5 ± 0.1 arbitrary units/g), but this difference was not significant. The relative content of P450scc per total corpus luteum was greater (P<0.01) in treated heifers (2.2 ± 0.2 arbitrary units) compared with control heifers (1.4 ± 0.2 arbitrary units). The relative content of P450scc per unit weight corpus luteum was also greater (P<0.05) in treated heifers (1.9 ±0.2 arbitrary units/g) compared with control heifers (1.2 ±0.1 arbitrary units/g). Relative content of 3ß-HSD per unit weight of corpus luteum tissue tended to be greater (Experiment 1: P=0.09; Experiment 2: P=0.72) in heifers treated with deslorelin (1.57 ± 0.15 arbitrary units/g) than in control heifers (1.49 ± 0.15 arbitrary units/g) but this was not significant. Relative content of 3ß-HSD per total corpus luteum likewise tended to be greater (P=0.20) in heifers treated with deslorelin (1.91 ± 0.25 arbitrary units) than in control heifers (1.51 ± 0.28 arbitrary units), but this also was not significant. In Experiment 3, stage of the oestrous cycle was synchronised in Brahman heifers, which were in random stages of the oestrous cycle, using a standard progestagen treatment. Heifers were then assigned to: Control (n=4), no treatment; or implanted with deslorelin (50 Lg/kg live weight/24 h) on different days of the oestrous cycle: D2 (n=4) implanted on Day 2; D4 (n=4) implanted on Day 4; D6 (n=4) implanted on Day 6; or D8 (n=4), implanted on Day 8 of the oestrous cycle. Ovarian follicle and corpus luteum status were monitored using ultrasonography for approximately 40 days. Heifers treated with deslorelin commencing on Day 2 of the oestrous cycle had follicles with a maximum diameter of 4.7 ± 0.3 mm at the time of treatment and did not ovulate or develop an accessory corpus luteum. The diameter of the largest follicle on Day 4 of the oestrous cycle was 6.5 ± 0.7 mm and two of four heifers ovulated and developed an accessory corpus luteum. The diameters of the two follicles that ovulated were 5.0 and 6.0 mm, while those that did not ovulate were 6.0 and 7.0 mm. On Day 6 of the oestrous cycle, each of the heifers had a 10 mm follicle that ovulated and developed an accessory corpus luteum. Heifers treated with deslorelin on Day 8 of the oestrous cycle had a relatively large follicle (8.63 ± 0.94 rum), but no heifers in this group ovulated. In 4 of 6 heifers that ovulated and developed an accessory corpus luteum, the spontaneous corpus luteum persisted for 11 to 19 days longer than the spontaneous corpus luteum in control heifers, even after the accessory corpus luteum had regressed. . The induced corpus luteum may have increased PGF₂receptors, or increased numbers of large luteal cells in the corpus luteum. Also, the presence of two corpora lutea might have produced sufficient oxytocin to cause downregulation of endometrial oxytocin receptors. The acute increase in plasma concentration of LH subsequent to implantation of heifers with deslorelin was consistent with previous findings. The rise in plasma LH was sufficient to induce ovulation of follicles of relatively small diameter (5 to 6 mm). This observation indicated that LH receptors sufficient for a response to a preovulatory LH surge are expressed in 6-7 mm follicles in Brahman (Bos indicus) heifers. Basal concentrations of plasma LH were consistently greater in heifers receiving deslorelin compared with untreated heifers. Increased basal LH in heifers treated with deslorelin was associated with greater concentrations of plasma progesterone. Because heifers treated with deslorelin would not have pulsatile secretion of LH, it could be concluded that basal secretion of LH is an important determinant of corpus luteum function and progesterone secretion in cattle. Heifers treated with deslorelin also had a larger corpus luteum and increased corpus luteum contents of StAR protein and P450scc. The latter findings are the first demonstration of concomitant increases in progesterone secretion, size of the corpus luteum and steroidogenic activity of luteal cells in a female mammal treated with GnRH agonist. Progesterone is required for the establishment and maintenance of pregnancy in cattle, and it is possible that treatment with a GnRH agonist may stimulate increased progesterone secretion sufficient to enhance conceptions to artificial insemination and embryo transfer in cattle.

History

Location

Central Queensland University

Additional Rights

I, the undersigned author of this thesis, understand that Central Queensland University make work available within the university library, and that it will be restrictions on access to this thesis. J. Desky Pitcher Statement of will this accessible to library users and other approved libraries. The thesis should not be copied, or closely paraphrased without the consent of the author, and written acknowledgment of the assistance gained from this work. Beyond this, I do not wish to place any Desley

Open Access

Yes

Era Eligible

No

Supervisor

Professor Michael J. D'Occhio ; Dr Paul Scott ; Dr Ray Rodgers

Thesis Type

Master's by Research Thesis

Thesis Format

By publication