Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH).

The effectiveness of different treatments with recombinant equine FSH to stimulate follicular growth, multiple ovulations and embryo production in seasonally anovulatory mares was evaluated. During mid-winter season (July-August in Argentina, South America) forty light breed donor mares, presenting follicles <10 mm in diameter and no CL at ultrasound examination (deep-anestrus), were randomly assigned (n = 10/group) to one of the following treatments: Group 1: twice daily intramuscular (IM) injections of 0.65 mg reFSH (AspenBio Pharma, CO), Group 2: once daily IM injection of 1.3 mg reFSH, Group 3: twice daily IM injection of 0.32 mg reFSH, and Group 4: once daily IM injection of saline (control). Treatment was administered until a follicle of 35 mm was observed or for a total period of 10 days. When the largest follicle reached ≥35 mm in diameter, treatment was discontinued and 2500 IU hCG was injected intravenously (IV) 36 h later. Mares receiving hCG were inseminated with fresh semen every 48 h until ovulation(s) were detected or one dose of frozen semen (250 × 106 motile sperm) after the first ovulation was detected. Eight days after first ovulation, transcervical embryo recovery was performed. Recovered embryos were non-surgically transferred to anovulatory estrogen/progesterone treated recipients and pregnancy diagnosed by ultrasonography 7, 14 and 21 days later. All mares receiving reFSH, but none receiving saline control, responded to the treatment with follicular growth. On average, 6.5 days of reFSH treatment were required for mares to develop follicles of ovulatory size (>35 mm). Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and in none of Group 4 (Control). Among ovulating mares, no differences in number of ovulations, number of embryos recovered, or pregnancy rates were observed among reFSH treatments. Of treated mares, 6, 7, and 5 produced embryos in Groups 1, 2, and 3, respectively. The average embryo recovery rate per ovulated mare was 88%. The average embryo recovery rate per ovulation was 43%. Overall, a 59% pregnancy rate was achieved. These results indicate that treatment with reFSH during deep anestrus results in follicular development, ovulation of fertile oocytes, and production of embryos that established viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily administrations, which allows for a simplified administration regimen.

Induction of ovarian activity and ovulation in an induced ovulator, the maned wolf (Chrysocyon brachyurus), using GnRH agonist and recombinant LH.

Assisted reproductive techniques, such as ovarian manipulation and artificial insemination, are useful for enhancing genetic management of threatened wildlife maintained ex situ. In this study, we used noninvasive fecal hormone monitoring to investigate (1) the influence of pairing with a male on endocrine responses of female maned wolves (Chrysocyon brachyurus) to a GnRH agonist (deslorelin) and (2) the efficiency of recombinant LH (reLH) on ovulation induction in females housed alone. Deslorelin (2.1 mg Ovuplant) was given to females that were either paired with a male (n = 4) or housed alone (n = 7); the implant was removed 7 to 11 days postimplantation. Three of seven singleton females were injected with reLH (0.0375 mg) on the day of implant removal, whereas the remaining females (n = 4) did not receive the additional treatment. Fecal samples were collected 5 to 7 days/wk from all females starting 11 days prior to hormone insertion until at least 70 days post implant removal for a total of 11 hormone treatment cycles. Fecal estrogen and progestagen metabolites were extracted and analyzed by enzyme immunoassay. Evidence of ovulation, demonstrated by a surge of estrogen followed by a significant rise in progestagen, occurred in all paired females. Three of the four singleton females that did not receive reLH treatment exhibited no rise in progestagen after an estrogen surge. All singleton females treated with reLH exhibited a rise in fecal progestagen after injection, indicating ovulation. In conclusion, deslorelin is effective at inducing ovarian activity and ovulation in paired female maned wolves; however, exogenous reLH is needed to induce ovulation in females housed alone. The findings obtained from this study serve as a foundation for future application of artificial insemination to enhance genetic management of this threatened species ex situ.

Induction of ovulation in seasonally anestrous mares under ambient lights using recombinant equine FSH (reFSH).

Traditionally, mares are put under artificial lights to advance the first ovulation of the year. The aim of the present study was to determine the efficacy of recombinant equine FSH (reFSH) in stimulating follicular development and advancing the first ovulation of the year in seasonally anestrous mares compared with anestrous mares given a placebo. Both groups of mares were housed under ambient light conditions. Sixty deep anestrous mares of light horse breeds (follicular diameters ≤ 20 mm in diameter and progesterone <1 ng/mL) were maintained under a natural photoperiod at three different sites: University of California, Davis, Colorado State University, and University of Kentucky Gluck Centre. Twenty mares at each site were randomly allocated to receive either 0.65 mg of reFSH (group A: treatment; n = 10) or a placebo (group B: control; n = 10) twice daily by im beginning on January 31. Treatment continued until one or more preovulatory follicles developed or up to a maximum of 15 days. Randomized treatments were blinded. Follicular development was closely monitored by transrectal ultrasonography. When the largest follicle reached ≥ 35 mm in diameter, reFSH treatment was discontinued and an injection of 2500 international units of hCG was administered iv 36 hours later to induce ovulation. Jugular blood samples were collected daily from all mares at University of California, Davis, and processed for LH, FSH, progesterone, estradiol-17ß, and immunoreactive-inhibin by RIA. All 30 mares receiving reFSH (group A) developed follicles ≥ 35 mm within 7.4 ± 1.6 days of treatment. Twenty-three of the 30 reFSH-treated mares (group A) ovulated within 72 hours after hCG administration. In contrast, mares in group B (placebo, control) did not exhibit significant follicular development and none ovulated within the 15-day observation period. Mares in group A had significantly higher plasma levels of FSH, estradiol-17ß, and immunoreactive-inhibin during treatment but did not exhibit a preovulatory LH surge. Mares administered reFSH returned to anestrus and spontaneously ovulated at a similar calendar date as control mares. These data indicate that reFSH was effective in stimulating the development of ovarian follicles and advancing the first ovulation of the year in seasonally anestrous mares under ambient lights but was not successful in inducing continued cyclicity.

S100A8/A9: a potential new diagnostic aid for acute appendicitis.

OBJECTIVES: Diagnosing acute appendicitis is a daunting clinical challenge, as there is no single test that reliably distinguishes acute appendicitis from other etiologies of acute abdominal pain. In this study, the authors examined whether circulating levels of S100A8/A9 could be useful as a marker to aid in the diagnosis of acute appendicitis. METHODS: Plasma samples from emergency department (ED) patients with acute abdominal pain (n=181) were tested using an immunoassay for S100A8/A9. RESULTS: The sensitivity and specificity for S100A8/A9 in diagnosing acute appendicitis were estimated to be 93% (95% confidence interval [CI]=81% to 97%) and 54% (95% CI=45% to 62%), respectively. Negative predictive value (NPV) was 96% (95% CI=89% to 99%), and positive predictive value (PPV) was 37% (95% CI=28% to 47%). Performance characteristics of elevated white blood cell (WBC) count were also estimated: sensitivity 63% (95% CI=47% to 76%), specificity 67% (95% CI=59% to 75%), NPV 86% (95% CI=78% to 91%), and PPV 36% (95% CI=26% to 47%). CONCLUSIONS: This is the first report exploring the relationship between circulating S100A8/A9 and acute appendicitis and establishes proof of concept for this biomarker as a diagnostic test for acute appendicitis. Further studies are indicated to optimize the use of this biomarker, in conjunction with other established approaches.

Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH).

To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was genetically fused to the amino end of the alpha subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH (n=5) or saline (n=3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure-function analyses and for breeding management in the horse.