Morphologic, Steroidogenic, and Transcriptomic Assessment of the Corpus Luteum in Holstein Cows after Spontaneous or Hormone-Induced Ovulation

Abstract

There is evidence that replacing the gonadotropin-releasing hormone (GnRH) with porcine luteinizing hormone (pLH) to synchronize ovulation prior to artificial insemination (AI) increased pregnancy per AI in dairy cows without affecting blood progesterone (P₄) concentrations. Whether morphologic, steroidogenic, and transcriptomic differences exist among corpora lutea (CL) formed after ovulation induced by GnRH and pLH is unclear. Our main objective, therefore, was to compare CL characteristics between GnRH- and pLH-induced CL. In 24 non-lactating Holstein cows, ovulations were spontaneous (Spont-Ov) or induced with 100 µg GnRH, 25 mg pLH, or 1 mg estradiol benzoate (EB), with CL excised 12 d after ovulation. In pLH- versus GnRH-treated cows, the duration of elevated LH (above baseline) was prolonged (10 versus 6 h, respectively, p < 0.01), but CL dimensions, pixel intensity of CL images, proportions of steroidogenic and non-steroidogenic luteal cells, and mean plasma LH did not significantly differ. Post-ovulation mean plasma P₄ (ng/mL) did not differ among Spont-Ov (3.0) pLH (3.1) or GnRH (3.0) cows but were lower in EB cows (2.0). In vitro P₄ concentration was greater in luteal explants of pLH-treated cows than in all other groups (combined means, 16.0 vs. 12.3 µg/mL, p < 0.02). Relative abundance of mRNA for oxytocin receptor (OXTR) was 2-fold higher (p < 0.01) in CL of pLH vs. GnRH cows and highest in Spont-Ov CL. In summary, pLH-treated cows had a longer LH peak, and greatest luteal tissue concentrations and in vitro production of P₄. We inferred that increased P₄ concentrations at the ovarian-uterine level in pLH-treated cows could have promoted embryo development and increased pregnancy per AI.

Keywords: GnRH; corpus luteum; estradiol benzoate; gene expression; histology; induced ovulation; luteal explants; porcine LH; progesterone.

Figure 1

Schematic timeline of the study. Thirty Holstein cows were treated with a 1.9 g progesterone intravaginal device (CIDR) for 5 d and 500 µg cloprostenol (PGF) at CIDR removal. Estrus was detected thrice daily for 3 d. On days 6 or 7 (Estrus = Day 0), all cows were treated twice with PGF, and randomly allocated to1 of 4 treatment groups to receive: 100 μg GnRH, 25 mg pLH, 1 mg estradiol benzoate (EB), or no treatment (Spont-Ov). Estradiol benzoate was administered 20 h after the first PGF, whereas pLH or GnRH was given 16 h later. Plasma concentrations of LH and progesterone (P₄) were determined in 28 and 24 cows, respectively. Transrectal ultrasonography (U/S) was used to determine CL dynamics, preovulatory follicle diameter, and ovulation.

Figure 2

Mean plasma progesterone (P₄) concentrations in non-lactating Holstein cows given 100 µg GnRH (n = 4), 25 mg pLH (n = 6), 1 mg estradiol benzoate (EB; n = 5), or no treatment (Spont-Ov, n = 4) following the administration of PGF. One cow that had a short estrous cycle after treatment with 100 µg GnRH was excluded. Plasma P₄ concentrations were affected by treatment and time (p < 0.01). Mean plasma P₄ in cows treated with 100 µg GnRH or 25 mg pLH did not differ from that in Spont-Ov cows. However, cows treated with 1 mg EB had lesser plasma P₄ concentrations. Treatment x time interaction tended (p = 0.09) to affect P₄ concentrations. The pooled SEM were 0.65, 0.60, 0.43, and 0.58 ng/mL for GnRH, pLH, EB and Spont-Ov, respectively. * GnRH, pLH and Spont-Ov > EB, p = 0.08. ** GnRH, pLH and Spont-Ov > EB, p < 0.05.

Figure 3

Mean plasma LH concentrations from cows treated with 100 μg of the gonadotropin-releasing hormone (GnRH; n = 5) or 25 mg of the porcine luteinizing hormone (pLH; n = 6). Mean LH concentrations did not differ by treatment (p > 0.05) but there was a time effect, and a treatment x time interaction (p < 0.01). The pooled SEM were 0.95 and 0.41 ng/mL for GnRH and pLH treatments, respectively. * GnRH > pLH, p < 0.01; ** pLH > GnRH, p < 0.01.

Figure 4

In vitro P₄ concentrations (µg/mL) from CL tissue and cultured media. Progesterone from CL tissue (pre-incubation) and cultured media (secreted P₄ in response to the addition of bovine LH at 0, 20 or 40 ng levels to the culture medium) in cows given GnRH (100 µg), pLH (25 mg), estradiol benzoate (EB; 1 mg), or no treatment (Spont-Ov) following PGF. ^a–c Bars without a common superscript within each cluster differ (p < 0.05). ^x,y p = 0.07.