Early conception factor lateral flow assays for pregnancy in the mare

The ECF™ lateral flow assay test is marketed to detect non-pregnancy in mares. The objectives of the present study were to determine the accuracy of the ECF test, the accuracy of the electronic reader accompanying the ECF test, and agreement between two human readers and the electronic reader. Serum samples were collected from anestrus, cycling but not inseminated, and inseminated mares, and were evaluated with the ECF™ test (EDP Biotech Company, Knoxville, TN, USA) at The Ohio State University and at the EDP Biotech Laboratory. Specificity ranged from 0.07 to 0.16, the negative predictive value ranged from 0.15 to 0.33, and accuracy ranged from 0.43 to 0.52. The electronic reader did not add improve the accuracy or predictive values of the test. Based on the electronic reader, 80.0% of the serum samples collected from the anestrus mares were false positives; Readers 1 and 2 had 60.0 and 33.3% false positives, respectively. For samples collected during the estrous cycle, 83.9% were false positives by the electronic reader, whereas Readers 1 and 2 had 43.7 and 26.4% false positives. We concluded that, regardless of whether the test strips were evaluated by a human or electronic reader, this assay was not accurate for determination of the non-pregnant mare.     

Antiluteogenic effects of serial prostaglandin F2α administration in cycling mares

A single dose of PGF2α does not consistently induce luteolysis in the equine CL until at least 5 days after ovulation, leading to the erroneous assumption that the early CL is refractory to the luteolytic effects of PGF2α. We hypothesized that serial administration of PGF2α in early diestrus would induce a return to estrus similar to mares treated with a single injection in mid-diestrus, and fertility of the induced estrus would not differ. The objectives of the study were to evaluate the effects of the 2 approaches as reflected by: (1) concentrations of plasma progesterone; (2) interovulatory and treatment-to-ovulation intervals; (3) the proportion of mares pregnant after artificial insemination. The study consisted of a balanced crossover design in which 10 reproductively normal Quarter Horse Mares were exposed to 2 treatments on 2 consecutive reproductive cycles. At detected ovulation (Day 0), mares were randomly allotted to 1 of 2 treatment groups: I, mid-diestrus treatment, administration of a single 10-mg dose of dinoprost tromethamine (PGF2α) im on Day 10; II, early diestrus treatment, administration of 10-mg PGF2α im twice daily on Days 0, 1, and 2 and once daily on Days 3 and 4. Mares in estrus and with a follicle 35 mm or greater in diameter were artificially inseminated with at least 2 billion motile sperm from a fertile stallion. Pregnancy was defined as detection of a growing embryonic vesicle on 2 consecutive examinations approximately 14 days after ovulation. Serial plasma samples were collected throughout the study period, and concentration of plasma progesterone was determined by RIA. A mixed-model ANOVA for repeated measures was used to analyze hormonal data. Interovulatory and treatment-to-ovulation intervals were compared by a paired t test and fertility by a McNemar chi-square analysis. All mares in group I underwent luteolysis after PGF2α administration denoted by mean (±SD) concentration of plasma progesterone of 0.25 ± 0.21 ng/mL detected 2 days after treatment. In group II, mean concentration of plasma progesterone remained below 1.0 ng/mL during treatment and until the onset of the next estrus. The mean interovulatory interval in group I was 18.5 ± 2.0 days compared with 13.1 ± 3.7 days in group II (P < 0.01). Treatment-to-ovulation intervals were 8.5 ± 2.0 days and 13.1 ± 3.7 days for groups I and II, respectively (P < 0.05). In both groups, 9 of 10 mares were pregnant (P = 1.0). Serial PGF2α administration beginning at ovulation consistently prevented luteal function in 10 of 10 mares in the present study without adversely affecting pregnancy rate of post-treatment cycles.     

Structural studies on equine chorionic gonadotropin

The amino acid sequence of the subunit of equine chorionic gonadotropin (eCG, also pregnant mare serum gonadotropin, PMSG) has been determined. Overlapping peptides from tryptic and chymotrypic digests were isolated by a two-dimensional peptide mapping technique and sequenced by the Edman procedure. The proposed amino acid sequence of eCG is: (**Denotes carbohydrate attachment points.) This sequence differs significantly from that proposed by Rathnamet al. (1978) for equine follitropin subunit; in particular, their sequence lacked the first fourteen residues.For the subunit we have placed in sequence 104 amino acid residues by direct sequence determination and peptide overlap procedures; in addition, 37 residues have been placed provisionally by homology with the human chorionic gonadotropin (hCG) sequence and composition and/or sequence data for the peptides isolated in the present studies. Difficulties in the procurement of the hormone have stalled completion of the -subunit amino acid sequence determination. The data now available indicate that eCG -subunit is highly homologous to hCG subunit and the subunits of luteinizing hormone from the pituitary gland of the several species so far described. The proposed partial sequence of eCG is:     

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2alpha induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2alpha on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares (n=8/group) were randomized into three groups: control (no treatment or biopsy), saline+biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2alpha+biopsy (5mg PGF2alpha at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline+biopsy group. At Hour 24 (12h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower (P<0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2alpha increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P<0.006), but decreased mRNA for LH receptor to 44% (P<0.05), steroidogenic acute regulatory protein to 22% (P<0.001), and aromatase to 43% (P<0.1) of controls. There was no difference in mRNA levels for PGF2alpha receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2alpha-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2alpha induced the synthesis of intraluteal PGF2alpha. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2alpha in mares.     

Pharmacologic application of native GnRH in the winter anovulatory mare,I: Frequency of reversion to the anovulatory state following ovulation induction and cessation of treatment

The continuous, subcutaneous infusion of native GnRH into seasonally anovulatory mares stimulates the synthesis and secretion of LH without pituitary refractoriness, offering opportunities to markedly accelerate the timing of ovulation within the operational breeding season. Herein, we tested the hypothesis that ovarian cycles induced in winter anovulatory mares using continuous administration of native GnRH for 28 days, beginning in either early February or early March (North America) would not revert to an anovulatory state after treatment withdrawal. Anovulatory mares received sham pumps (control) or native GnRH (100 μg/h) for 28 days beginning from February 2 or 3 (GnRH-Feb) or March 2 or 3 (GnRH-Mar). Mean concentrations of LH were five- to seven-fold greater during February in the GnRH-Feb group compared with control and GnRH-Mar groups through February and ending on March 2 or 3. However, concentrations of LH returned to the winter baseline within 3 to 11 days after pump removal and all GnRH-Feb mares failed to remain cyclic after treatment withdrawal. Correspondingly, during March, concentrations of LH in the GnRH-Mar group were greater (P < 0.001) than in the control and GnRH-Feb groups during the 28-day treatment period. Follicular growth and frequency of ovulation (6/10 GnRH-Feb; 9/10 GnRH-Mar, 1/11 controls, respectively) were greater (P < 0.01) in GnRH-treated mares. Ovulatory cycles continued in five of nine GnRH-Mar mares that ovulated, with interovulatory intervals of 15 to 24 days; whereas, three of nine mares had extended (33–42 days) interovulatory intervals and one of nine mares had a persistent CL after cessation of treatment. In summary, continuous administration of native GnRH for 28 days, beginning in early February or March, elevated circulating LH adequately to stimulate follicular growth and ovulation up to 60 days earlier than in untreated controls. However, if continuous, subcutaneous infusion of GnRH is selected as the only pharmacologic or managerial intervention, and mares are not pregnant, treatment must be continued at least until the end of March. This will improve the likelihood of a normal interovulatory interval after treatment withdrawal.     

The cryoprotective effect of trehalose supplementation on boar spermatozoa quality

The objective was to compare the reproductive performances associated with the first (Cycle-1), second (Cycle-2), and mid-season (MS-Cycle) ovulations of the breeding season in donor mares that were treated with equine-FSH (eFSH) in the early vernal transition. Mares (n = 15) kept under ambient light were examined ultrasonographically per-rectum starting January 30. When an ovarian follicle ≥25 mm in diameter was detected, twice daily eFSH treatments were initiated. The eFSH treatments ceased when a follicle ≥35 mm was detected, and 36 h later hCG was administered. Thereafter, mares were artificially inseminated every 48 h until ovulation (Day 0). Trans-cervical embryo recovery attempts were performed on Day 8, and subsequently PGF2α was administered. Equine FSH was not administered in the subsequent estrous cycles. In Cycle-2 and in the MS-Cycle, hCG was administered when a follicle ≥35 mm was detected; breeding, embryo recovery, and PGF2α administration, were similar to Cycle-1. Mares had an untreated estrous cycle (no treatment or breeding) between Cycle-2 and the MS-Cycle. All mares developed follicle(s) ≥35 mm after 4.9 ± 0.6 days of eFSH treatment, and subsequently ovulations occurred; mean (95% CI) interval from treatment initiation to ovulation was 7.9 (6.5–9.3) days. The number of preovulatory follicles (≥30 mm) at the time of hCG administration (Cycle-1: 2.2 ± 0.3 compared with Cycle-2: 1.0 ± 0 compared with MS-Cycle: 1.1 ± 0.1 follicles), and the number of ovulations (2.5 ± 0.4 compared with 1.0 ± 0 compared with 1.1 ± 0.1 ovulations) were greater (p < 0.05) in Cycle-1. Nevertheless, mean embryo numbers did not differ among cycles (0.8 ± 0.2 compared with 0.5 ± 0.1 compared with 0.5 ± 0.1 embryo/mare). On average, embryo morphology grade was less (p < 0.05) in Cycle-1 as compared to non-eFSH cycles (combined Cycle-2 and MS-Cycle). This impaired embryo quality could be due to a seasonal effect, or negative effect of the eFSH treatment, which was possibly related to alterations in the hormonal environment (estradiol-17β and progesterone). A prolonged IOI (>21 days) was recorded in 7 of 15 mares following the Cycle-1 ovulation, but not subsequently. In conclusion, eFSH treatment of vernal transitional donor mares stimulated ovulation within only few days of treatment, and the following embryo recovery rate was at least as good as in the subsequent estrous cycles; however, on average, embryos were morphologically impaired. In subsequent estrous cycles in the breeding season, ovulations, embryo recovery rates, and embryo variables did not appear to be negatively affected; however, the first inter-ovulatory interval of the breeding season was prolonged in approximately half of the mares.     

Early effects of equine FSH (eFSH) treatment on hormonal and reproductive parameters in mares intended to carry their own pregnancy

Superovulatory treatment may potentially increase the embryo recovery rate and the per-cycle pregnancy rate in normal or subfertile mares that are managed properly. However, some studies suggest a possible negative effect of superovulatory treatment on ovarian follicular maturation and embryo viability. Objectives of the present study were to investigate the early effects of eFSH treatment in reproductively normal mares in terms of: folliculogenesis, pregnancy rate, early embryonic development, reproductive tract parameters (tone and edema), and serum estradiol-17β and progesterone concentrations. Reproductively sound mares (n = 26) were evaluated daily by transrectal palpation and ultrasonography. Five days after spontaneous ovulation, mares were randomly assigned to one of two treatment groups. In the eFSH group, mares (n = 16 estrous cycles) were administered eFSH twice daily; beginning when a follicle ≥20 mm was detected, and continuing until at least one follicle reached a diameter of ≥35 mm. PGF2α was administered 2 days following initiation of eFSH therapy, and hCG was administered approximately 36 h after cessation of eFSH therapy. In the control group, mares (n = 26 estrous cycles) were administered PGF2α 7 days after spontaneous ovulation, and hCG when a follicle ≥35 mm was detected. All mares were bred with fresh semen, monitored for ovulation (Day 0), and evaluated for pregnancy on Days 11–16. Serum estradiol-17β and progesterone concentrations were analyzed using radioimmunoassay on the Day of hCG administration, and Days 8, 11 and 16. Mares treated with eFSH had more follicles ≥30 mm at the time of hCG administration (2.6 ± 0.4 compared with 1.1 ± 0.1; P < 0.01), and more ovulations (2.3 ± 0.5 compared with 1.1 ± 0.3; P < 0.01). However, pregnancy rates were not significantly different between groups (50%; 8/16 compared with 62%; 16/26). Mean overall daily growth rate of embryonic vesicles from Day 11 to 16 was not statistically different between the two groups (3.3 ± 0.3 compared with 3.7 ± 0.1 mm/day) (P = 0.2); however, was more variable (P < 0.01) in the eFSH group (95%CI: 2.6–3.8 mm/day) than in the control group (95%CI: 3.5–3.9 mm/day). Administration of eFSH modified the reproductive tract variables and serum concentrations of progesterone and estradiol-17β on the days that oocyte maturation, fertilization, and early embryonic development are expected to occur. These alterations may be related to the greater incidence of non-ovulatory follicles (25% compared with 0%), fewer embryos per ovulation rate (0.3 ± 0.1 compared with 0.6 ± 0.1), and the lesser than expected pregnancy rates in the eFSH-treated mares.     

The effect of hormone treatments (hCG and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: A field study

The association between use of hormone treatments to induce estrus and ovulation and the incidence of hemorrhagic anovulatory follicles (HAFs) was studied in a mixed population of mares (Equus caballus) during two breeding seasons in a commercial breeding clinic. Mares treated with cloprostenol (CLO) were more likely to develop HAFs than were mares with spontaneous cycles (P < 0.001) or those treated with human chorionic gonadotropin alone (P = 0.08). There was no significant effect of season on the incidence of HAFs. The mean (±SEM) interval from CLO treatment to beginning of HAF development was 6.1 ± 0.5 d. Age of mares with HAF cycles was not different (12 ± 1.3 yr; P > 0.05) from that of mares with ovulatory cycles (10.5 ± 1.5 yr).     

Effect of type of semen, time of insemination relative to ovulation and embryo transfer on early equine embryonic vesicle growth as determined by ultrasound

Embryonic vesicle growth in the mare is easily monitored by ultrasound. Apart from pregnancy diagnosis, assessment of the embryonic vesicle in practice is also useful to evaluate its viability. Although subject to individual variation, embryo growth rate follows a constant pattern in the early stages of development in relation to embryonic age. Previous studies have shown a significant effect of some factors routinely used in practice, such as post-ovulation insemination and embryo transfer, on embryonic growth and the time in which the vesicle is first detected. This study attempts to confirm previous results in different settings and characterise the causes for this delay in growth. A total of 159 pregnancies from different mating protocols: (1) pre-ovulation natural mating, (2) pre-ovulation natural mating and transfer into recipient mares, (3) post-ovulation natural mating, and (4) post-ovulation AI with frozen/thaw spermatozoa were evaluated ultrasonographically from day 12 to 19 of pregnancy and vesicle diameters recorded. Regression analysis between embryonic vesicle diameters and embryonic ages was performed for each group and mean vesicle diameter at different age periods among groups were tested for statistical difference with a general linear model of variance. There was no significant difference between groups 1 and 2 (P = 0.73) or between groups 3 and 4 (P = 0.71). However both pre-ovulation groups (1 and 2) had larger vesicle diameters (P < 0.000) at any embryonic age analysed than either of the post-ovulation groups (3 and 4). In conclusion, post-ovulation inseminations produced pregnancies with smaller vesicle diameters equivalent to approximately 1 day's growth.