Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)


Animal Science

First Advisor

George Perry


beef, reproduction


In cattle, estradiol is responsible for modulating many mechanisms involved in successful reproduction. Specifically estradiol is the primary signal for the initiation of standing estrus (Allrich, 1994), and cattle expressing estrus prior to fixed time AI (FTAI) have been reported to have increased preovulatory estradiol concentrations (Perry and Perry, 2008) and increased pregnancy success compared to animals that did not exhibit estrus (Richardson et al., 2016). Gonadotropin releasing hormone, classically, stimulates estradiol production via the two-cell two-gonadotropin hypothesis (Fortune and Quirk, 1988), and GnRH administered systemically in small doses (5 μg) has been reported to elicit an LH pulse similar to a physiological pulse (Ginther et al., 1996). In a study where multiple small doses (5 μg) of GnRH were administered following CIDR removal, concentrations of estradiol were increased (Larimore et al., 2016). These studies demonstrate that estradiol production from ovarian follicles could be stimulated with systemically administered GnRH. In addition, to the classical pathway, GnRH receptor mRNA has been characterized in bovine granulosa cells (Ramakrishnappa et al., 2003), and GnRH has been reported to have local stimulatory and inhibitory actions on steroidogenesis within the ovary (Sharpe, 1982; Janssens et al., 2000). The role of GnRH in bovine follicles as it pertains to estradiol production and/or the regulation of estradiol production has not been well characterized. Therefore, the objective of the subsequent studies were to investigate the relationship of GnRH and its impact on estradiol production and other reproductive parameters by conducting field trials where GnRH was administered systemically (Chapters 3 and 4), as well as the local abundance of GnRH-I and GnRH-II mRNA within granulosa cells of bovine antral follicles (Chapter 5). In Chapter 3, beef cows and heifers (n = 1620) were synchronized using the 7-day CIDR protocol, and randomly assigned to one of three treatments (0, 5, and 10 μg of a GnRH analog at CIDR removal). Animals were visually observed for estrus and inseminated following detection in estrus. Interval to estrus was calculated for each animal that exhibited estrus (INTERVAL 1). Animals that did not exhibit estrus were given 100 μg of GnRH at the time of AI and their interval to estrus was designated at 120 h (INTERVAL 2). There was an effect of age (P < 0.01) and a treatment by age interaction (P = 0.05) on INTERVAL 1. Heifers had a shorter interval to estrus than cows (50 h vs 54 h respectively). Furthermore, heifers given 5 μg of GnRH tended to have a shorter interval to estrus (P = 0.07; 47 ± 1.4 h) compared to 0 μg (50 ± 1.5 h) and did have a shorter interval compared to 10 μg (P < 0.01; 52 ± 1.5 h). There were no differences among treatments in interval to estrus among cows (P ≥ 0.34). When animals that did not exhibit estrus were included in the analysis at 120 h there was no treatment by age interaction (P = 0.49). This is likely due to the fact that treatment (P < 0.01), but not age (P = 0.96) or treatment by age (P = 0.74) influenced expression of estrus, with 5 μg tending to have more animals in estrus (P = 0.10; 79 ± 4%) compared to 0 μg (74 ± 5%), and 10 μg having fewer animals in estrus compared to either other treatment (P < 0.04; 68 ± 6%). Estrus (P < 0.01) and age (P < 0.01) influenced pregnancy success with heifers having greater pregnancy success compared to cows (49 ± 5% vs 38 ± 4%, respectively) and animals exhibiting estrus having greater pregnancy success compared to animals that did not exhibit estrus (57 ± 4% vs 32 ± 4%, respectively). However, there was no difference in pregnancy success between treatments among animals that exhibited estrus (P > 0.30). Among animals that did not exhibit estrus 0 μg had increased pregnancy success (P ≤ 0.05; 40 ± 6%) compared to 5 μg and 10 μg which did not differ (27 ± 6% and 29 ± 5%, respectively). This experiment demonstrated that small doses of GnRH following CIDR removal can shorten the interval to estrus and increases the percentage of animals exhibiting estrus prior to AI. In Chapter 4, beef cows and heifers (n = 247) were synchronized using the 7-day CO-Synch + CIDR protocol and were assigned to one of three treatment groups [0 μg GnRH at CIDR removal (0 μg); 10 μg GnRH at CIDR removal (10 μg), or 5 μg at CIDR removal plus 5 μg 12 h later (5 + 5 μg)]. Animals were visually observed for estrus and artificially inseminated 55 (heifers) or 60 (cows) hours following CIDR removal (FTAI). Blood samples were collected beginning at CIDR removal and every 12 hours until time of AI. Concentrations of estradiol were not influenced by treatment (P = 0.66) or a treatment by time interaction (P = 0.87), but were influenced by time (P < 0.0001). Estradiol concentrations increased from CIDR removal to 48 hours after CIDR removal and then were decreased at the time of AI. Expression of estrus was not influenced by age (P = 0.31), or treatment (P = 0.60), however, there was a treatment by age interaction (P < 0.01) for expression of estrus. For animals administered the 5 + 5 μg treatment, expression of estrus was increased (P < 0.01) among heifers compared to cows (84 ± 3% vs 70 ± 3%, respectively). Pregnancy success was influenced by estrus (P < 0.01), treatment (P = 0.02), and a treatment by estrus (P < 0.01) interaction, but was not influenced by age (P = 0.91), treatment by age (P = 0.83), or treatment by estrus by age (P = 0.94). Animals that exhibited estrus had increased pregnancy success compared to animals that did not exhibit estrus (67 ± 2% vs 41 ± 7% respectively). Animals administered the 5 + 5 μg treatment had increased pregnancy success (P ≤ 0.05; 69 ± 6%) compared to animals administered the 0 μg and 10 μg treatments which did not differ (53 ± 6% vs 41 ± 8%, respectively). Pregnancy success was similar (P > 0.27) between animals that did and did not exhibit estrus for animals administered the 0 μg and 5 + 5 μg treatments. For animals administered the 10 μg treatment, conception rates were decreased (P < 0.01) among animals that did not exhibit estrus compared to animals that exhibited estrus. This study demonstrated that the 5 + 5 μg treatment of GnRH following CIDR removal, when implementing a fixed-time AI protocol, positively influenced conception rates. In Chapter 5, beef cows/heifers were synchronized using the CO-Synch protocol and artificially inseminated. On day 16 after insemination animals were transported to a local abattoir. Following slaughter ovaries were collected and all follicles were classified as small (< 5 mm), medium (5 to 10 mm), or large (> 10 mm). Follicles were aspirated to collect follicular fluid and granulosa cells. Follicles were pooled by size within animal (n = 23, 16, and 18 for small, medium, and large, respectively). Follicular fluid concentrations of estradiol were determined by radioimmunoassay. Total cellular RNA was extracted from the granulosa cells and RT-PCR was performed to determine relative abundance of mRNA for GnRH-I, GnRH-II, and GAPDH. Data were analyzed using the mixed procedure in SAS. Follicle size influenced concentration of estradiol. Large follicles had increased estradiol (P < 0.0001) compared to small and medium follicles (18,626 ± 2,650 vs 1,270 ± 2,307 and 8,925 ± 2,763 pg/mL, respectively). There was no difference (P = 0.31) in relative abundance of GnRH-I mRNA among small, medium, or large follicles (3.8 ± 0.78, 3.40 ± 0.85, and 1.95 ± 0.94; respectively). Relative abundance of GnRH-II mRNA was influenced by follicle size (P < 0.05), with greater abundance in small follicles (40.97 ± 9.27) compared to medium (6.32 ± 11; P = 0.02) or large (7.85 ± 11.11; P = 0.02) follicles. However, there was no difference (P = 0.92) in relative abundance between medium and large follicles. When follicles were classified by concentration of estradiol, follicles with the lowest 25% of estradiol had decreased (LOWE2; P < 0.0001) concentrations of estradiol (434 ± 1,074 pg/mL) compared to the middle 50% (MIDE2; 7,029 ± 961 pg/mL) which was decreased (P < 0.0001) compared to the greatest 25% (HIGHE2; 46,423 ± 2,025 pg/mL). LOWE2 had greater (P < 0.01; 5.37 ± 0.67) abundance of GnRH-I mRNA compared to MIDE2 and HIGHE2 which did not differ (1.85 ± 0.55 and 1.23 ± 1.65, respectively). Relative abundance of GnRH-II mRNA was greater (P < 0.01) in LOWE2 (42.33 ± 9.56) compared to MIDE2 (5.98 ± 8.90). HIGHE2 was similar (P ≥ 0.12) to the other two groups (8.90 ± 19.12). Results from this study demonstrate a relationship between granulosa cell GnRH-I and II mRNA abundance with estradiol concentrations within the follicle, where decreased GnRH-I and GnRH-II may be playing a role in increased estradiol production. These three experiments demonstrate GnRH directly impacting estradiol, and/or reproductive performance of beef cows and heifers through interval to estrus, expression of estrus, and increasing conception rates when used with FTAI protocols. The mechanisms by which GnRH is having its actions remains to be elucidated.


Includes bibliographical references (pages 95-114).



Number of Pages



South Dakota State University


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