Evaluation of progesterone deficiency as a cause of fetal death in mares with experimentally induced endotoxemia

The role of decreased luteal activity in embryonic loss after induced endotoxemia was studied in mares 21 to 35 days pregnant. Fourteen pregnant mares were treated daily with 44 mg of altrenogest to compensate for the loss of endogenous progesterone secretion caused by prostaglandin F2 alpha (PGF2 alpha) synthesis and release following intravenous administration of Salmonella typhimurium endotoxin. Altrenogest was administered daily from the day of endotoxin injection until day 40 of gestation (group 1; n = 7), until day 70 (group 2; n = 5), or until day 50 (group 3; n = 2). In all mares, secretion of PGF2 alpha, as determined by the plasma 15-keto-13,14-dihydro-PGF2 alpha concentrations, followed a biphasic pattern, with an initial peak at 30 minutes followed by a second, larger peak at 105 minutes after endotoxin injection. Plasma progesterone concentrations decreased in all mares to values less than 1 ng/ml within 24 hours after endotoxin injection. In group 1, progesterone concentrations for all mares were less than 1 ng/ml until the final day of altrenogest treatment. In 6 of 7 mares in group 1, the fetuses died within 4 days after the end of treatment, with progesterone concentrations less than 1 ng/ml at that time. In the mare that remained pregnant after the end of treatment, plasma progesterone concentration was 1.6 ng/ml on day 41 and increased to 4.4 ng/ml on day 44. In group 2, all mares remained pregnant, even though plasma progesterone concentrations were less than 1 ng/ml in 4 of 5 mares from the day after endotoxin injection until after the end of altrenogest treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of Medroxyprogesterone Acetate in Suppression of Estrus in Cycling Mares

The effects of compounded medroxyprogesterone acetate (MPA) on follicular activity and estrous behavior were evaluated. Eighteen cycling mares were assigned to one of three treatment groups. Mares in the MPA group (n = 6) were injected intramuscularly with 1,600 mg MPA (week 1), then 400 mg weekly for the next 5 weeks. Saline mares (n = 6) were injected intramuscularly weekly for 6 weeks. Altrenogest mares (n = 6) received 10 mL orally daily for 7 weeks. Mares were teased daily for 60 days and categorized as displaying estrous, diestrous, or neutral behavior. Transrectal ultrasound examinations were performed three times weekly, or daily when a 30-mm follicle was identified, until ovulation. Blood samples were harvested weekly for analysis of progesterone concentration and daily from days 14 to 23 for analysis of luteinizing hormone (LH) concentration. Mares treated with saline or MPA showed normal intervals of diestrus and estrus during the study. All altrenogest mares showed behavioral diestrus during treatment. All mares in the saline and MPA groups showed normal follicular development and ovulations. No altrenogest mares ovulated during treatment; four mares returned to estrus and resumed normal follicular development after treatment ceased. Progesterone analyses agreed with transrectal ultrasonographic ovarian activity for all mares. LH levels were lower for altrenogest-treated mares compared with MPA-treated and saline-treated mares during the treatment period. In conclusion, compounded MPA at dose rates and intervals used in this study was not effective in suppression of estrus, follicular development, or LH secretion in mares.     

Effect of altrenogest‐treatment of mares in late gestation on adrenocortical function,blood count and plasma electrolytes in their foals

Reasons for performing study: Mares with compromised pregnancies are often treated with altrenogest to prevent abortion. However, there is only limited information about effects on the foal when altrenogest treatment is continued during final maturation of the fetus. Objectives: To determine effects of altrenogest treatment during late gestation in mares on maturity, haematology changes, adrenocortical function and serum electrolytes in their newborn foals. Methods: Six mares were treated with altrenogest (0.088 mg/kg bwt) once daily from Day 280 of pregnancy until foaling and 7 mares served as controls. Results: Foals born to altrenogest‐treated mares had a significantly lower neutrophil/lymphocyte ratio on the first day after birth than control foals (P<0.05). Basal plasma cortisol concentrations immediately after birth were higher in foals of altrenogest‐treated mares than in control foals (P<0.05). Cortisol release in response to exogenous adrenocorticotropic hormone (ACTH) ‐ except for higher values 15 min after ACTH injection in foals of altrenogest‐treated mares on Day 1 ‐ revealed no differences in adrenocortical function between the groups of foals. Plasma potassium concentration in foals from altrenogest‐treated mares compared to control foals was significantly lower immediately after birth (P<0.05) and plasma ionised calcium concentration was significantly lower 3 h after birth (P = 0.01). Conclusions and potential relevance: Altrenogest treatment of pregnant mares prolonged labour had no major effects on adrenocortical function in foals. A reduced neutrophil/ lymphocyte ratio in these foals may suggest either immunomodulatory effects of altrenogest or dysmaturity of the foals.     

Estradiol and progesterone concentrations resulting from the administration of an exogenous hormone regimen in diestrous embryo transfer recipients

Estradiol and progesterone concentrations were evaluated from diestrous embryo transfer recipient mares (5 to 14 days post-ovulation) which were treated with an exogenous hormone regimen. Upon detection of the donor mare's ovulation (0 hours), 10 mg PGF_2α was given to the recipient mare; at 12, 24 and 36 hours 20 mg estradiol cypionate; at 48 hours, 500 mg progesterone in oil and then 22 mg altrenogest at 60, 72 and 96 hours. Altrenogest (22 mg/day) was continued until end of the trial (detection of a fetal heart beat). Embryos were transferred non-surgically 6 or 7 days after the start of treatment.Plasma samples were evaluated over three periods; period 1-between recipient mare ovulation and prior to PGF_2α period 2-between PGF and embryo transfer and period 3-post-transfer. During periods 2 and 3, estradiol was higher (P<.05) for mares which were 10 to 14 days post-ovulation (late diestrous) as compared to mares which were 5 to 9 days post ovulation (mid-diestrous) when treatment began. Progesterone concentrations were higher (P<.05) for the mid-diestrous mares in the same periods. The pregnancy rate was higher for the late diestrous mares than the mid-diestrous mares (58% (7/12) vs 10% (1/10)). However, no difference (P>.05) was detected in estradiol or progesterone in the late diestrous mares which were pregnant or open. During period 2, estradiol was higher (P<.05) in the pregnant than open mares. Whereas, during period 3, progesterone was higher (P<.05) in the open mares.These data suggest that estradiol is important for the establishment of pregnancy in the mare. Furthermore, hormone treatment developed in this study appears to have some potential in synchronization of diestrus mares to be used as embryo recipients.     

Progestins in Mid- to Late-Pregnant Mares

Two experiments were conducted to determine when a placental source of progestin was sufficient for maintaining pregnancy in the mare. In the first study, embryos were transferred into ovariectomized mares and pregnancy was maintained with altrenogest^a Altrenogest treatment was terminated at either day 100 (n=6) or day 150 (n=6). Twelve ovarian-intact mares were assigned to a second experiment on day 100 of gestation. On day 160 of gesta- tion, these mares were assigned to one of three treatments: 1) ovariectomy on day 160 and given altrenogest to day 200 (n=4); 2) ovariectomy on day 180 and given altrenogest to day 250 (n=4); or 3) ovariectomy on day 200 and given altrenogest to day 300 (n=4). Blood samples were collected every 2 weeks from all mares in both experiments from day 100 to parturition and assayed for concentrations of pro- gestins. Pregnancy loss from day 100 to parturition was not different among groups in either experiment. Serum con- centrations ofprogestins in ovary-intact mares were greater (P<0.05) than those in ovariectomized pregnant mares until day 130, after which they were similar. Serum con- centrations of progestins in the ovariectomized pregnant mares rose gradually from day 100 until near parturition. Serum concentrations of progestins in the ovary-intact pregnant mares declined from day 100 to day 157, did not vary significantly from day 157 to day 245, then rose until near parturition. Serum concentrations ofprogestins tended to decrease the sixth day prior to parturition. From these     

Effect of repeated administration of oxytocin during diestrus on duration of function of corpora lutea in mares

OBJECTIVE: To determine whether IM administration of exogenous oxytocin twice daily on days 7 to 14 after ovulation blocks luteolysis and causes prolonged function of corpora lutea (CL) in mares. DESIGN: Prospective study. ANIMALS: 12 mares. PROCEDURES: Beginning on the day of ovulation (day 0), jugular blood samples were collected every other day until day 40 for determination of progesterone concentration. On day 7, mares (n = 6/group) were treated with saline (0.9% NaCl) solution (control group) or oxytocin. Beginning on day 7, control mares received 3 mL of sterile saline solution every 12 hours, IM, and oxytocin-treated mares received 60 units of oxytocin every 12 hours, IM, through day 14. Mares were considered to have prolonged CL function if progesterone concentration remained > 1.0 ng/mL continuously through day 30. RESULTS: The proportion of mares with prolonged CL function was significantly higher in the oxytocin-treated group (6/6), compared with the control group (0/6). All control mares underwent luteolysis by day 16, at which time their progesterone concentrations were < 1.0 ng/mL. In contrast, all 6 oxytocin-treated mares maintained progesterone concentrations > 1.0 ng/mL continuously through day 30. CONCLUSIONS AND CLINICAL RELEVANCE: IM administration of 60 units of oxytocin twice daily on days 7 to 14 after ovulation was an efficacious method of inhibiting luteolysis and extending CL function in mares. Disrupting luteolysis by administering exogenous oxytocin during diestrus appears to be a plausible and practical method of long-term suppression of estrus in mares.     

Reproductive response of mares after treatment with progestogens with and without the additional of estradiol

A study involving 60 light-horse mares was conducted both to evaluate the response of mares to injectable progester- one or altrenogest and to determine ifestradiol in combination with either progestogen provided any added benefit. Treatments were initiated at either early estrus, late estrus, early diestrus, mid-diestrus or late diestrus in order to assess the effect of stage of cycle at onset of treatment. Within each of these stages of the cycle, mares were randomly assigned to 1 of 4 treatments: 150 mg progesterone injected i.m. (P); 150 mg progesterone + 10 mg estradio11713 injected i.m. (P+); .044 mg altrenogest per kg body weight orally (A); and .044 mg per kg body weight orally plus 10 mg estradiol 1713 i.m. (A+). All treatments were given daily for 7 days with 10 mg PGFaCt given on day 7 to all mares. The number of mares ovulating by day 14 after treatment (N=15/group) was 13, 7,11 and 8 forA, A+, P and P+, respectively. The response of mares to progesterone and altrenogest was similar. Fewer (Pì0.05) mares given combined steroid treatments ovulated within 14 days (15 of 30) than those given progestogen treatments. Stage of cycle had no affect (Pì0.05) on response of mares ovulating within 14 days or after 14 days of treatment. Mares that ovulated within 14 days of treatment had larger foUieles after progestogen treatment than those not ovulating by 14 days.     

Reproductive traits, lactation and foal growth in mares fed altrenogest

Lactating mares were assigned as controls or fed altrenogest (.044 mg.kg body wt-1.d-1) for 15 d after foaling. Mares (n = 6) fed altrenogest were inseminated during the first estrus after treatment and mares (n = 6) in the control group were inseminated during the second postpartum estrus. Ovulation during the estrus in which mares were inseminated occurred 26 +/- 1 d postpartum for treated mares and 36 +/- 1 d postpartum for control mares. The percentage of mares conceiving was not different for control (67%) and alternogest-treated (100%) mares. No differences were observed in tone and size of the uterus or size of the ovulatory follicle between treated and control groups. Uterine cultures and biopsies collected on d 7 and 15 postpartum were similar between treatment and control groups in bacterial populations or endometrial epithelial cell height. Blood was collected on d 7, 11, 15, 19 and 23 postpartum, and concentrations of estradiol-17 beta in serum were determined by radioimmunoassay. Mean concentrations of estradiol-17 beta across days were 10 +/- .8 and 12 +/- .6 pg/ml for control and treated mares, respectively. Concentrations of serum estradiol-17 beta were higher (P less than .05) in treated mares on d 23 postpartum. Daily milk yields, determined by the weigh-suckle-weigh method, and milk composition were similar between treatment groups on each collection day. Altrenogest can be used to predictably delay estrus in the postpartum mare without altering fertility, yield and composition of milk, or foal growth.     

Follicle Development during Luteal Phase and Altrenogest Treatment in Pigs

Synchronization of the oestrous cycle of gilts using altrenogest treatment has been found to increase ovulation rate. The current experiment investigated if the increase in ovulation rate after altrenogest treatment is related to increased follicle size at the end of altrenogest treatment compared with late luteal phase follicles. Crossbred gilts (n = 15) received altrenogest during 18 days [20 mg Regumate (Janssen Animal Health, Beerse, Belgium)], starting 5-7 days after onset of first oestrus. Control gilts (n = 15) did not receive altrenogest. At days 10-12 of the oestrous cycle [i.e. in the presence of corpora lutea (CL)], average follicle development was 2.51 +/- 0.20 mm (assessed with ultrasound) in altrenogest-treated gilts and 2.58 +/- 0.16 mm in control gilts (p > 0.10). During the last days of altrenogest treatment (i.e. when CL had gone into regression), average follicle size had increased to 3.01 +/- 0.31 mm (p < 0.05). Subsequent ovulation rate was 16.6 +/- 1.7 in altrenogest treated gilts and 15.1 +/- 1.2 in control gilts (p < 0.05). Altrenogest treatment resulted in increased follicle size after regression of the CL, showing that suppression of follicle growth by altrenogest alone is less severe than suppression by endogenous progesterone (either with or without altrenogest). Altrenogest treatment also resulted in a higher ovulation rate. However, it is unclear if the increased follicle size and higher ovulation rate after altrenogest treatment are causally related, as the relation between the two on an animal level was not significant.     

Evaluation of Injectable Sustained Release Progestin Formulations for Suppression of Estrus and Ovulation in Mares

Thirty-one mares were used in an experiment to evaluate the effectiveness of three sustained-release injectable formulations of altrenogest and one formulation of medroxyprogesterone acetate (MPA) for long-term suppression of estrus and ovulation. Luteolysis was induced by injection of prostaglandin-F_2α (Lutalyse) on day 0 (6th day after the previous ovulation) and was immediately followed by treatment with 1) no injection (controls; n = 7), 2) 1.5 mL of an altrenogest solution in sustained-release vehicle (LA 150, 1.5 mL; 225 mg altrenogest; n = 6), 3) 3 mL (450 mg altrenogest) of the same solution (n = 6), 4) 500 mg altrenogest in lactide-glycolide microparticles suspended in 7-mL vehicle (MP 500; n = 6), or 5) 1.0 g MPA as a 5-mL suspension. Mares were checked for estrus daily, and their ovaries scanned every other day until a 25-mm or greater follicle was detected, after which they were scanned daily. Control mares returned to estrus an average of 3.9 days after Lutalyse administration; all the single-injection altrenogest formulations increased (P < .05) the days to return to estrus, with the greatest increase occurring in mares receiving MP 500. Return to estrus was not affected by MPA treatment. Time of ovulation was determined by serial ultrasound scans and confirmed by daily plasma luteinizing hormone (LH) and progesterone concentrations. Control mares ovulated an average of 8.8 days after Lutalyse administration. Treatment with 1.5 or 3 mL of LA 150 increased (P < .05) the mean days to ovulation to 16.5 and 21.2 days, respectively; MP 500 increased (P < .05) the days to ovulation to 33.5 days. Administration of MPA did not affect (P > .1) days to ovulation relative to control mares. The MP 500 treatment provided long-term suppression of estrus and ovulation and could prove useful for that purpose. Treatment with the LA 150 solutions provided shorter-term suppression, and a relatively tight grouping of the individual mares around the mean days to ovulation; these one-shot formulations could be useful for synchronizing ovulation in cyclic mares and inducing normal estrous cyclicity in vernal transitional mares exhibiting erratic, anovulatory estrous periods.     

Safety of altrenogest in pregnant mares and on health and development of offspring

Fifty-one light-horse mares were utilized to evaluate the safety of an oral progestin, altrenogest, administered throughout gestation on: gestation length, embryonic and fetal loss, periparturient events, health and development of offspring, and future reproductive capabilities of the mares. Pregnancies were established by inseminating mares with 250 × 10⁶ progressively motile spermatozoa from the same stallion every other day throughout estrus or by non-surgical transfer of embryos. Mares were randomly assigned to 1 of 2 treatments upon confirmation of pregnancy on day 20: 1) controls, 2 ml of neobee oil orally per 44.5 kg of body weight; and 2) treated, 2 ml of altrenogest dissolved in neobee oil at a concentration of 2.2 mg/ml orally per 44.5 kg of body weight. Treatments were administered daily from day 20 to 320 of gestation.There were no significant differences between treatment groups for duration of gestation, placental weight, time to placental expulsion and incidence of retained placental membranes. Number of female foals born from altrenogest treated mares (14 of 23) was greater (P<.05) than the number from untreated control mares (4 of 16). Female foals born from altrenogest treated mares had larger clitori (P<.05) than those from control mares. Times to sternal recumbency, standing and nursing were similar for the 2 groups (P>.05). Body weight and height at withers, heart girth circumference and length and width of cannon were measured at time of birth and at 2, 4, 6, 8, 12 and 16 weeks of age. Measurements did not differ (P>05) between treated and control foals for any development parameters.Beginning on day 20 postpartum, mares were teased daily. During estrus, mares were inseminated every other day with 250 × 10⁶ motile spermatozoa. Teasing and/or insemination was continued for 2 cycles or until mares were 35 days pregnant. The number of mares pregnant after 1 cycle and after 2 cycles of insemination was similar (P>.05) for treated and control mares. Nineteen of 21 treated mares and 15 of 16 control mares were pregnant after 2 cycles of insemination. Number of cycles per pregnancy was similar (P>.05) for treated and control mares (1.37 vs 1.13) as was number of days mares exhibited estrus (6.30 vs 6.13). Number of inseminations per cycle did not differ (P>.05) between treated and control mares (2.92 vs 3.00). In summary, there was no effect of treatment with altrenogest from day 20 to 320 of gestation on periparturient events, viability and growth of offspring and subsequent reproductive performance of mares.     

Use of progesterone in microspheres for maintenance of pregnancy in mares.

Administration of progesterone in poly(d-,l-lactide) microspheres was used to maintain pregnancy in mares after luteolysis was induced by treatment with prostaglandin F2 alpha at day 14 of pregnancy. Mares were given vehicle only (control, n = 6) or 0.75 g (n = 7), 1.5 g (n = 8), or 2.25 g (n = 5) of microencapsulated progesterone at days 12 and 22 of pregnancy. Serum progesterone concentrations were determined daily, and pregnancy was evaluated by transrectal ultrasonography on alternate days. Significantly (P less than 0.05) more mares given 1.5 or 2.25 g of progesterone (6 of 8 and 4 of 5 mares, respectively), but not those given 0.75 g (3 of 7 mares), maintained pregnancy through day 32, compared with control mares (0 of 6). Progesterone concentrations decreased significantly (P less than 0.025) in all groups after administration of prostaglandin F2 alpha at day 14, and significant (P less than 0.05) effects of time and treatment on progesterone concentrations were found between days 12 and 22, and 22 and 32. Although treatment with 1.5-g and 2.25-g doses of microencapsulated progesterone improved maintenance of pregnancy, compared with that of vehicle-treated controls, administration of 2.25 g of microencapsulated progesterone appeared to be most efficacious in maintenance of pregnancy during the study interval.     

Superovulation in cycling mares using equine follicle stimulating hormone (eFSH)

Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Other potential clinical applications include improving pregnancy rates from frozen semen, treatment of subfertility in stallions and mares, and induction of ovulation in transitional mares. The objective of this study was to evaluate the efficacy of purified equine follicle stimulating hormone (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) in inducing superovulation in cycling mares. In the first experiment, 49 normal, cycling mares were used in a study at Colorado State University. Mares were assigned to 1 of 3 groups: group 1, controls (n = 29) and groups 2 and 3, eFSH-treated (n = 10/group). Treated mares were administered 25 mg of eFSH twice daily beginning 5 or 6 days after ovulation (group 2). Mares received 250 (of cloprostenol on the second day of eFSH treatment. Administration of eFSH continued until the majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting on day 5 or 6. The treatment regimen was identical to that of group 2. Mares in all 3 groups were bred with semen from 1 of 4 stallions. Pregnancy status was determined at 14 to 16 days after ovulation.In experiment 2, 16 light-horse mares were used during the physiologic breeding season in Brazil. On the first cycle, mares served as controls, and on the second cycle, mares were administered 12 mg of eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time human chorionic gonadotropin (hCG) was administered. Mares were inseminated on both cycles, and embryo collection attempts were performed 7 or 8 days after ovulation.Mares treated with 25 mg of eFSH developed a greater number of follicles (35 mm) and ovulated a greater number of follicles than control mares. However, the number of pregnancies obtained per mare was not different between control mares and those receiving 25 mg of eFSH twice daily. Mares treated with 12 mg of eFSH and administered either hCG or deslorelin also developed more follicles than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles than control mares, whereas the number of ovulations from mares receiving eFSH followed by deslorelin was similar to that of control mares. Pregnancy rate for mares induced to ovulate with hCG was higher than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of the controls. Overall, 80% of mares administered eFSH had multiple ovulations compared with 10.3% of the control mares.In experiment 2, the number of large follicles was greater in the eFSH-treated cycle than the previous untreated cycle. In addition, the number of ovulations during the cycle in which mares were treated with eFSH was greater (3.6) than for the control cycle (1.0). The average number of embryos recovered per mare for the eFSH cycle (1.9 ± 0.3) was greater than the embryo recovery rate for the control cycle (0.5 ± 0.3).In summary, the highest ovulation and the highest pregnancy and embryo recovery rates were obtained after administration of 12 mg of eFSH twice daily followed by 2500 IU of hCG. Superovulation with eFSH increased pregnancy rate and embryo recovery rate and, thus, the efficiency of the embryo transfer program.

Introduction

Induction of multiple ovulations or superovulation has been an elusive goal in the mare. Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates.[1 and 2] Superovulation also has been suggested as a critical requirement for other types of assisted reproductive technology in the horse, including oocyte transfer and gamete intrafallopian transfer. [2 and 3] Unfortunately, techniques used successfully to superovulate ruminants, such as administration of porcine follicle stimulating hormone and equine chorionic gonadotropin have little effect in the mare. [4 and 5]The most consistent therapy used to induce multiple ovulations in mares has been administration of purified equine pituitary gonadotropins. Equine pituitary extract (EPE) is a purified gonadotropin preparation containing approximately 6% to 10% LH and 2% to 4% FSH.[6] EPE has been used for many years to induce multiple ovulations in mares [7, 8 and 9] and increase the embryo recovery rate from embryo transfer donor mares. [10] Recently, a highly purified equine FSH product has become available commercially.The objectives of this study were to evaluate the efficacy of purified eFSH in inducing superovulation in cycling mares and to determine the relationship between ovulation rate and pregnancy rate or embryo collection rate in superovulated mares.

Materials and methods

Experiment 1

Forty-nine normally cycling mares, ranging in age from 3 to 12 years, were used in a study at Colorado State University. Group 1 (control) mares (n = 29) were examined daily when in estrus by transrectal ultrasonography. Mares were administered an implant containing 2.1 mg deslorelin (Ovuplant, Ft. Dodge Animal Health, Ft. Dodge, IA) subcutaneously in the vulva when a follicle 35 mm in diameter was detected. Mares were bred with frozen semen (800 million spermatozoa; minimum of 30% progressive motility) from 1 of 4 stallions 33 and 48 hours after deslorelin administration. The deslorelin implants were removed after detection of ovulation.[11] Pregnancy status was determined at 14 and 16 days after ovulation.Group 2 mares (n = 10) were administered 25 mg of eFSH (Bioniche Animal Health USA, Inc., Athens, GA) intramuscularly twice daily beginning 5 or 6 days after ovulation was detected. Mares received 250 g cloprostenol (Estrumate, Schering-Plough Animal Health, Omaha, NE) intramuscularly on the second day of eFSH treatment. Administration of eFSH continued until a majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Mares were subsequently bred with the same frozen semen used for control mares, and pregnancy examinations were performed as described above.Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting 5 or 6 days after ovulation and were administered 250 μg cloprostenol on the second day of treatment. Mares were randomly selected to receive either a deslorelin implant (n = 5) or 2500 IU of human chorionic gonadotropin (hCG) intravenously (n = 5) to induce ovulation when a majority of follicles reached a diameter of 35 mm. Mares were bred with frozen semen and examined for pregnancy as described above.

Experiment 2

Sixteen cycling light-horse mares were used during the physiologic breeding season in Brazil. Reproductive activity was monitored by transrectal palpation and ultrasonography every 3 days during diestrus and daily during estrus. On the first cycle, mares were administered 2500 IU hCG intravenously once a follicle 35 mm was detected. Mares were subsequently inseminated with pooled fresh semen from 2 stallions (1 billion motile sperm) daily until ovulation was detected. An embryo collection procedure was performed 7 days after ovulation. Mares were subsequently administered cloprostenol, and eFSH treatment was initiated. Mares received 12 mg eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time hCG was administered. Mares were inseminated and embryo collection attempts were performed as described previously.

Statistical analysis

In experiment 1, 1-way analysis of variance with F protected LSD was used to analyze quantitative data. Pregnancies per ovulation were analyzed by x² analysis. In experiment 2, number of large follicles, ovulation rate, and embryo recovery rate were compared by Student,'s t-test. Data are presented as the mean S.E.M. Differences were considered to be statistically significant at p < .05, unless otherwise indicated.

Results

In experiment 1, mares treated with 25 mg eFSH twice daily developed a greater number of follicles 35 mm in diameter (p = .001) and ovulated a greater number of follicles (p = .003) than control mares (Table 1). However, the number of pregnancies obtained per mare was not significantly different between the control group and the group receiving 25 mg eFSH (p = .9518). Mares treated with 12 mg eFSH and administered either hCG or deslorelin to induce ovulation also developed more follicles 35 mm (p = .0016 and .0003, respectively) than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles (p = .003) than control mares, whereas the number of ovulations for mares receiving eFSH followed by deslorelin was similar to that of control mares (p = .3463). Pregnancy rate for mares induced to ovulate with hCG was higher (p = .0119) than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of controls (p = .692). Pregnancy rate per ovulation was not significantly different between control mares (54.5%) and mares treated with eFSH followed by hCG (52.9%). The lowest pregnancy rate per ovulation was for mares stimulated with 25 mg eFSH and induced to ovulate with deslorelin. The mean number of days mares were treated with 25 mg or 12 mg of eFSH was 7.8 ± 0.4 and 7.5 ± 0.5 days, respectively. Overall, 80.0% of mares administered eFSH had multiple ovulations compared with 10.3% of control mares.     

Effect of exogenous progesterone administration on luteal sensitivity to PGF during the early development of the corpus luteum in mares and cows

The objective of this study was to determine the effect of exogenous progesterone administration at ovulation and during the early development of the CL, on its future sensitivity to a single administration of PGF2a in mares and cows. Horse Retrospective reproductive data from an equine clinic in the UK during three breeding seasons were used. Mares were divided into: control group, cycles with single ovulations; double ovulation group cycles with asynchronous double ovulations; and PRID group: cycles with single ovulations and treatment with intravaginal progesterone device (CIDR) immediately after the ovulation. All mares were treated with d‐cloprostenol (PGF) at either: (i) 88 hr; (ii) 96 hr; (iii) 104 hr; or (iv) 112 hr after the last ovulation. Cattle A total of nine non‐lactating Holstein cows were used. All cows were administered PGF14 d apart and allocated to one of two groups control group GnRH was administered 56 hr after the second PGF administration. CIDR group CIDR was inserted at the same time of GnRH administration. All cows were administered PGF at 120 hr post‐ovulation. The complete luteolysis rate of mares with double ovulation (66.7%) and those treated with exogenous progesterone (68.4%) was significantly higher than the rate of mares with single ovulation (35.6%) at 104 hr. In the cow, however, the treatment with CIDR did not increase the luteolytic response in cows treated at 120 hr post‐ovulation. In conclusion, the degree of complete luteolysis can be influenced by increasing the concentration of progesterone during the early luteal development in mares.     

The Effects of Oral Altrenogest on Hormonal,Testicular, and Behavioral Profiles of Two-Year-Old Stallions

Altrenogest is frequently administered to young stallions in the equine industry as an off-label use to suppress sexual/aggressive behavior. In this study, 2-yr-old Quarter Horse stallions in early-performance training were administered altrenogest (0.044 mg/kg BW daily) to determine the effects on sexual/aggressive behavior, testicular parameters, and steroid hormone profiles. Horses were randomly assigned to treatment (n = 5) and control (n = 5) groups. The treatment group was administered a daily oral dose of 0.044 mg/kg BW daily for 67 d; the control group was given a daily oral sham dose of corn oil for 67 d. No significant differences were found between treatment groups in BW or body condition scores. Altrenogest had no effect on any behavioral parameters or seminal parameters measured. Averages of total scrotal width (TSW) were lower (P<0.03) for treatment animals at the end of treatment period (d 67). By d 157 mean TSW for both treatment and control stallions were not different. Both were significantly higher (P<0.03) than pretrial values at d 157. Comparisons of testicular weight (d 157) between control and treatment groups were not different. Histological analysis of testicular tissue revealed no significant difference for the average number of spermatids per seminiferous tubule. Altrenogest treatment reduced serum estrogen levels (estradiol 17-β) by d 67 (P<0.02); however, estrogen levels returned to pre-treatment values after a 90-d recovery. Serum testosterone values were not affected by treatment. Further research is needed to determine dose and age effects of altrenogest in the stallion.     

Efficacy of short-term administration of altrenogest to postpone ovulation in mares

Estrogen from a growing follicle stimulates the preovulatory surge of luteinizing hormone (LH) while progesterone (P) is known to suppress LH. The possibility exists that administration of P, in the presence of an ovulatory follicle, would sufficiently suppress LH and, therefore, delay ovulation. The objective of this research was to elucidate the potential for oral administration of altrenogest (17-Allyl-17β-hydroxyestra-4,9,11-trien-3-one) to postpone ovulation of a preovulatory follicle (35 mm) for approximately two days. Fourteen light-horse mares, ranging in age from two to 19 years, were randomly assigned to one of three treatments (A-.044 mg/kg BW altrenogest for two days; B-.088 mg/kg BW altrenogest for two days; and C- no altrenogest). Mares began treatment when a 35-mm or greater follicle was observed via real-time transrectal ultrasonography. Both number of days until ovulation and follicular maintenance differed between treated and control mares. Number of days until ovulation was increased (P<.05) for mares in treatment A when compared with the control mares. Follicular diameter maintenance, a measurement of follicular diameter throughout treatment, also increased (P<.05) for mares in treatment A when compared with the control mares. Mean LH concentration was not different between mares treated with altrenogest at either treatment dose when compared with the control mares. Pregnancy rates and embryonic vesicle size change were also measured to determine potential effects of altrenogest administration. No differences (P>.05) were found in either characteristic.Short-term administration of altrenogest increased the number of days to ovulation. Further study is warranted to prove conclusively that altrenogest increases follicular maintenance, alters the preovulatory LH surge, and has no detrimental effects upon reproductive efficiency.     

Progestagen supplementation during early pregnancy does not improve embryo survival in pigs

Progesterone supplementation during early pregnancy may increase embryo survival in pigs. The current study evaluated whether oral supplementation with an analogue of progesterone, altrenogest (ALT), affects embryo survival. A first experiment evaluated the effect of a daily 20-mg dosage of ALT during days 1-4 or 2-4 after onset of oestrus on embryo survival at day 42 of pregnancy. A control group (CTR1) was not treated. The time of ovulation was estimated by transrectal ultrasound at 12-h intervals. Altrenogest treatment significantly reduced pregnancy rate when start of treatment was before or at ovulation: 25% (5/20) compared to later start of treatment [85% (28/33)] and non-treated CTR1 [100% (23/23)]. Altrenogest treatment also reduced (p < 0.05) number of foetuses, from 14.6 ± 2.6 in CTR1 to 12.5 ± 2.5 when ALT started 1-1.5 days from ovulation and 10.7 ± 2.9 when ALT started 0-0.5 days from ovulation. In a second experiment, sows with a weaning-to-oestrous interval (WOI) of 6, 7 or 8-14 days were given ALT [either 20 mg (ALT20; n = 49) or 10 mg (ALT10; n = 48)] at day 4 and day 6 after onset of oestrus or were not treated (CTR2; n = 49), and farrowing rate and litter size were evaluated. Weaning-to-oestrous interval did not affect farrowing rate or litter size. ALT did not affect farrowing rate (86% vs 90% in CTR2), but ALT20 tended to have a lower litter size compared with CTR2 (11.7 ± 4.1 vs 13.3 ± 3.1; p = 0.07) and ALT10 was intermediate (12.3 ± 2.9). In conclusion, altrenogest supplementation too soon after ovulation reduces fertilization rate and embryo survival rate and altrenogest supplementation at 4-6 days of pregnancy reduces litter size. As a consequence, altrenogest supplementation during early pregnancy may reduce both farrowing rate and litter size and cannot be applied at this stage in practice as a remedy against low litter size.     

Efficacy of altrenogest administration to postpone ovulation and subsequent fertility in mares

A recent report suggested administration of altrenogest during the follicular phase could postpone ovulation. Based on these results, two questions were generated. We first hypothesized that by initiating a altrenogest treatment earlier in the estrous cycle, a greater and/or more consistent delay in ovulation would result. Second, we hypothesized that exposure to elevated progestin concentrations might alter viability of the ovulatory follicle and oocyte. The focus of the first experiment was to determine if initiation of altrenogest treatment at different stages of the estrous cycle would yield a more predictable time to ovulation, whereas the second experiment was designed to determine whether mares receiving altrenogest during estrus had compromised fertility. In the first experiment thirty mares of mixed light breed, ranging in age from 5-15 years, were randomly assigned to one of three groups. The two treated groups received altrenogest (0.088 mg/kg of body weight) for two days once a follicle of 30 or 35 mm in diameter was detected. Control mares were not treated. Mares treated with altrenogest whether initiated at the detection of a 30 or 35 mm follicle demonstrated similar (P>.05) day to ovulation interval when adjusted to 35 mm (5.4 and 5.6 days, respectively). Both treated groups demonstrated a delayed interval (P<.05) when compared to control (3.9 days). Thirty-six mares of similar breed and age, were randomly assigned to two groups for use in the second experiment. All mares were monitored daily via transrectal ultrasonography from the time a 35 mm or greater follicle was detected until ovulation. Treated mares received daily doses of altrenogest (0.088 mg/kg of body weight) for two days once a follicle of 35 mm or greater was detected. Control mares received no treatment. Fertility data were collected from mares inseminated every other day with 500 million motile spermatozoa from one of two stallions with proven fertility. Pregnancy data were collected via transrectal ultrasonography at days 12, 14 and 16 post-ovulation. Ovulation data were collected from 27 control cycles and 26 treated cycles. Contrary to previous reports and Experiment 1, no difference (P=0.35) was noted between groups with respect to days to ovulation. Control mares averaged 4.14 days and treated mares averaged 4.7 days to ovulation from initial detection of a 35 mm follicle. Fertility data were also similar (P=0.8) between control and treated mares (66.6% and 61.5% per cycle, respectively). Interestingly, a greater number (P=0.017) of treated cycles (5/26) resulted in follicular regression than did control cycles (0/27). While these data suggest that this dosage of altrenogest may not postpone ovulation, it did appear related to increased incidence of follicular regression. Fertility was unaffected, however, in those mares that ovulated. Further studies are needed in which initiation at different stages of estrus and different doses of altrenogest are used.     

Effectiveness of a two-dose regimen of prostaglandin administration in inducing luteolysis without adverse side effects in mares

Our objectives were to determine whether repeated administration of prostaglandin F2alpha (PGF2alpha) to simulate the endogenous mode of secretion would be more effective than a single injection in inducing luteolysis and enable use of smaller doses less likely to cause adverse side effects. The main study comprised 43 dioestrous mares, who were given im. either a single 10 mg dose of natural PGF2alpha (n = 22) or 2 doses of 0.5 mg PGF2, 24 h apart (n = 21). The intensity of side effects was assessed in 8 dioestrous mares given 5, 1.5, 0.5 or 0 mg PGF2alpha in consecutive cycles. Two doses of 0.5 mg PGF2alpha 24 h apart caused lysis of the corpus luteum in all mares, whether this was determined from a fall in plasma progesterone concentrations or reproductive tract/behavioural changes; and when 10 mg PGF2, was given, the corpus luteum was lysed in 17 of 22 mares i.e. a lower proportion (P = 0.0485). A single dose of 0.5 mg PGF2a was no more effective than saline in inducing luteolysis.The intensity of side effects of PGF2alpha increased with dose. Although the 0.5 mg dose was no more likely than saline to cause sweating or muscle spasms, it raised plasma cortisol concentrations and prevented the decline in heart rate seen after saline. We conclude that a 2 dose regimen of administration increases the luteolytic efficacy of PGF2alpha and thereby provides a way to minimise adverse side effects.     

Embryo Transfer in Anovulatory Recipient Mares Treated with Estradiol Benzoate and Long-Acting Progesterone

This study aimed to prepare anovulatory mares in anestrus or in the transitional period as embryo recipients. Ninety embryo-recipient mares were divided into two groups (G). G1 (n = 45) comprised animals in anestrus or in the transitional period; these animals were treated for 3 days (D) with 5, 3, and 2 mg of estradiol benzoate (intramuscular) on D0 (day of the donor's ovulation), D1, and D2 (after ovulation), respectively, followed by weekly application of 400 mg of long-acting progesterone (intramuscular) from D3 after ovulation (donor) until the 120th day of gestation. G2 (n = 45) comprised mares with normal estrous cycles. Plasma levels of progesterone (P₄) were measured on days D1, D2, D8, and D14. Sixty percent of the animals in G1 and 71.1% in G2 (P > .05) completed the pregnancy. On D8, there was no difference in P₄ levels between G1 and G2 animals, but there was a difference in P₄ levels on D14 (P < .05). It was concluded that anovulatory mares in anestrus or in the transitional period could be used as embryo recipients. The protocol was efficient and also considered an appropriate alternative to prepare the uterine environment for embryo transfer; long-acting progesterone administration kept P₄ levels high enough to maintain pregnancy until the 120th day and provided recipients during the time of the year when fewer mares were cycling and ovulating.