Untreated Mares Research Articles

Immunocontraception Limits Foal Production in Free-Roaming Feral Horses in Nevada

We determined contraceptive effectiveness and duration of effect after porcine zona pellucida (PZP) vaccination in free-roaming feral horses in Nevada. We captured, freeze-branded, treated and subsequently released 132 adult feral mares. Treatment consisted of 1 or 2 injections (1 month apart) of vaccine emulsion consisting of aqueous PZP and Freund's complete adjuvant (Sigma, St. Louis, Mo.; FCA; inj. 1) or Freund's incomplete adjuvant (FIA; inj. 2). We gave controls saline and Freund's adjuvant as above. Among 1-injection mares, we gave one group a single dose of raw antigen, and we gave the other group raw antigen plus a second dose of antigen sequestered in controlled-release microspheres. We also studied 63 untreated mares. We administered inoculations in December 1992, and monitored the mares via fecal analysis for pregnancy and via helicopter and ground survey for foal production through September 1994. We determined pregnancy via measurement of estrone sulfate, progesterone metabolites and total estrogens, in fresh feces collected from the ground. Among antigen-treated mares, reproductive success across 1 year was 4.5% (2-inj.), 20.0% (1-inj. + microspheres) and 28.6% (1-inj., no microspheres). Reproductive success in placebo (55.0%) and untreated (53.9%) mares was significantly greater than in treated groups. The following year, without further treatment, reproductive success was 44.0% (2-inj.), and 54.5% (untreated) and these values were not different. Data from other groups were insufficient for comparison. The results of this study confirm the feasibility of capturing large numbers of mares in the'field for treatment and demonstrate: (1) 1-year contraceptive efficacy of several PZP vaccine protocols, (2) greater effectiveness in 2-injection protocols than in 1-injection protocols, and (3) a return to fertility in the second year after vaccination.

Effects of cervical dilation and intrauterine infusions on the timing of oviductal transport of equine embryos

Equine embryos spend 5 to 6 d in the oviduct before entering the uterus as expanded blastocysts, and cannot be consistently collected nonsurgically until Day 7. Technologies such as cryopreservation and embryo splitting, which are most successful with embryos at the morula or early blastocyst stage, have not been used in mares because equine morulae and early blastocysts are located in the oviduct and cannot be recovered nonsurgically. These experiments test the hypothesis that transport of equine embryos through the oviduct can be hastened by cervical dilation or by acute, sterile endometritis induced by intrauterine oyster glycogen treatment. Cervical dilation with or without intrauterine infusion of 0.5 ml PBS on Day 4 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for mares with cervical dilation or cervical dilation plus PBS infusion vs mares receiving no treatments (0 of 5 and 0 of 5 vs 0 of 10, respectively). Intrauterine infusions of 40 ml of 1% oyster glycogen or 40 ml of PBS on Day 3 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for oyster glycogen- or PBS-treated vs untreated mares (2 of 12 and 3 of 11 vs 0 of 10, respectively). Cervical and uterine treatments on Day 3 or Day 4 and uterine lavages on Day 5 decreased (P < 0.05) Days 11 to Day 15 pregnancy rates compared with that of untreated mares. Day 11 to Day 15 pregnancy rates were 1 of 5 for mares with Day 4 cervical dilation and Day 5 uterine lavage, 1 of 5 for mares with Day 4 PBS infusion and Day 5 uterine lavage, 2 of 12 for mares with Day 3 oyster glycogen infusion and Day 5 uterine lavage, and 3 of 11 for mares with Day 3 PBS infusion and Day 5 uterine lavage vs 7 of 10 for mares that received no treatment or lavage. Cervical and uterine manipulations on Day 3 or 4 and uterine lavage on Day 5 appeared to decrease pregnancy rates by Days 11 to 15. The results of these experiments do not support the hypothesis that cervical dilation or uterine infusion hasten oviductal transport, since neither cervical manipulation nor transcervical infusion of oyster glycogen or PBS into the uterus significantly hastened the rate of embryo transport into the uterus.

Equine immunocontraception using porcine zona pellucida: A new method for remote delivery and characterization of the immune response

Equine immunocontraception using porcine zona pellucida: A new method for remote delivery and characterization of the immune response

Observations on the use of progesterone intravaginally and of deslorelin STI in acyclic mares for induction of ovulation

Summary In this clinical study, 29 acyclic mares with inactive ovaries were treated with an intravaginal device containing 1.9 g progesterone (CIDR-B) for 10 to 12 days. Mares which formed a follicle >30 mm after CIDR-B removal received a subcutaneous implant containing 1.5 mg or 2.2 mg deslorelin, a potent GnRH analog. Twelve mares were treated during the breeding season (BS), 17 during autumn and winter anestrus (AS). During fall-winter anestrus, 77 untreated patient mares served as contemporary controls. Due to the uniformity of response, data were pooled. Twenty-seven out of 29 mares responded to CIDR-B treatment with follicle growth; 22 mares were treated with deslorelin when follicles had reached 38 mm within 2.6+2.3 days after CIDR-B withdrawal and 20 mares ovulated within 2.77+1.66 days. Fifteen mares were inseminated and 10 (66.7%) became pregnant at first breeding. Half of the mares treated during AS with CIDR-B only, and not with deslorelin ovulated; in all other of those mares, the preovulatory-size follicle regressed eventually.

Embryo transfer in anoestrous recipient mares: attempts to reduce altrenogest administration period by treatment with pituitary extract

SummaryGroups of ovariectomised and anoestrous mares were treated with altrenogest and used as recipients for embryo transfer. Progesterone concentrations in all ovariectomised, pregnant mares increased only after 85 days of pregnancy, but in 2 out of 4 anoestrous pregnant mares progesterone increased between Days 40 and 50 of pregnancy. Follicular growth and luteinised follicles suggested that altrenogest could be withdrawn earlier. To test whether exogenous LH treatment could stimulate earlier onset of ovarian progesterone secretion, anoestrous, recipient pregnant mares treated with altrenogest were treated with LH (25 mg CEG (crude equine gonadotrophin) im daily (N=5). Two of the 4 control mares ovulated at Days 58–60 and plasma progesterone increased. The other 2 mares did not ovulate. In the mares treated with CEG, altrenogest treatment was withdrawn in 3 on Days 45, 50 and 55 respectively. One mare aborted on Day 54 when endogenous progesterone was still very low and there was no indication of ovulation and/or luteinisation in the 5th mare. There was no significant difference in increasing progesterone concentrations between CEG‐treated mares and untreated mares, suggesting that such treatment in anoestrous recipient mares is relatively inefficient.

An immunohistochemical study of bovine antral follicles, with special attention to non-atretic follicles with and without atypical granulosa cells.

Inhibin and oxytocin were immunohistochemically demonstrated in all non-atretic and light-atretic follicles > 2 mm from untreated and pregnant mare's serum gonadotrophin (PMSG)-treated heifers and cows. Immunostaining for luteinizing hormone (LH) and oestradiol was observed in all non-atretic follicles > 4 mm, but only in follicles from PMSG-treated cows. Inhibin and oestradiol immunoreactivity was restricted to the granulosa. Oxytocin and LH immunoreactivity was visualized in both the theca interna and the granulosa. Within the granulosa, LH immunoreactivity was mainly present in cells that were located near the basement membrane. Normal granulosa cells differed from atypical granulosa cells (AGCs) with respect to their ability to bind LH and oestradiol. It is concluded that immunostaining for alpha-inhibin, oxytocin, oestradiol and LH cannot be used as a marker of follicle quality to discriminate between non-atretic follicles with AGCs and non-atretic follicles without AGCs in mid-luteal bovine ovaries.

Long-term effects of porcine zonae pellucidae immunocontraception on ovarian function in feral horses (Equus caballus).

Ten feral mares free-roaming in Maryland, USA, were inoculated with porcine zonae pellucidae (PZP) protein before the breeding season for three consecutive years (1988-90). Ovarian function was monitored for 51 days during the peak of the breeding season after the third annual PZP inoculation, in seven of these mares and in four untreated control mares, by means of urinary oestrone conjugates and nonspecific progesterone metabolites. None of the ten inoculated mares became pregnant in 1990, compared with 55% of 20 control mares, which included two of the four monitored for ovarian function. Three of the untreated mares demonstrated apparent normal ovarian activity, characterized by preovulatory oestrogen peaks, concurrent progesterone nadirs at ovulation, breeding activity, and luteal-phase progesterone increases after ovulation. Two of the seven monitored PZP-treated mares demonstrated ovulatory cycles that did not result in conception. One was pregnant as a result of conception in 1989 and demonstrated a normal, late-gestation, endocrine profile. The remaining four PZP-treated mares revealed no evidence of ovulation, and urinary oestrogen concentrations were significantly depressed. The experiments indicated that (i) a third consecutive annual PZP booster inoculation is greater than 90% effective in preventing pregnancies in mares and (ii) three consecutive years of PZP treatment may interfere with normal ovarian function as shown by markedly depressed oestrogen secretion.

Use of gonadotropin-releasing hormone for hastening ovulation in transitional mares.

Natural GnRH and its analog have potential for hastening ovulation in mares. A study was conducted to evaluate the efficacy of a GnRH agonist given either as an injectable or s.c. implant for induction of ovulation in mares. Forty-five seasonally anestrous mares (March) were assigned to one of three groups (n = 15/group): 1) untreated controls; 2) i.m. injection of the GnRH agonist buserelin at 12-h intervals (40 micrograms/injection for 28 d or until ovulation) and 3) GnRH agonist administered as a s.c. implant (approximately 100 micrograms/24 h for 28 d). Six mares per group were bled on d 0, 7, 14 and 21 after injection or insertion of implant. Samples were taken at -1, -.5 and 0 h and at .5, 1, 1.5, 2, 4, 6 and 8 h after GnRH. Additional daily samples were drawn for 28 d after injection or until ovulation. Samples were assayed for concentration of LH and FSH. Progesterone concentrations were determined in samples collected on d 4, 6 and 10 after ovulation. Number and size of follicles and detection of ovulation were determined by ultrasonography. Number of mares induced to ovulate within 30 d was 0 of 15, 7 of 15 and 9 of 15 for groups 1, 2 and 3, respectively. During treatment, follicle sizes were smaller for mares in group 3 (implant). The LH response to GnRH agonist (area under curve) was similar among groups at d 0 but was greater (P less than .05) for mares in group 3 on d 7 and 14 and groups 2 and 3 on d 21 than for controls. A similar pattern was detected for peak concentrations of LH after GnRH on d 0, 7, 14 and 21. Daily concentrations of LH remained low in untreated control mares compared with GnRH-treated mares throughout the sampling period. Concentrations of LH for mares in group 3 that ovulated were elevated greatly above those for group 2 mares, whereas concentrations of FSH were similar in both treatment groups prior to ovulation.

The effect of constant delivery of GnRH on the episodic secretion of equine LH

Abstract Based on the results of two experiments, constant delivery of gonadotropin releasing hormone (GnRH) via osmotic pumps to progestin-pretreated anovulatory mares elevated circulating concentrations of luteinizing hormone (LH) to levels observed in estrous mares. Furthermore, the episodic nature of LH concentrations were still detected for the six days that mares were continuously administered GnRH. However, there was an apparent 22% decrease in the frequency of LH peaks per hour while mean peak lengths were 27% longer for mares administered a constant delivery of GnRH than for untreated mares.

Effect of exogenous progesterone on pregnancy rates after surgical embryo transfer in mares

A completely randomized experimental design was used to investigate the effect of supplemental progesterone on pregnancy rates of recipient mares. Every other recipient mare received daily 200 mg progesterone in oil beginning the day of surgical embryo transfer and lasting until either Day 120 of pregnancy or until pregnancy failure was confirmed by ultrasound. Progesterone supplementation did not affect pregnancy rate (P > 0.05). Overall, embryos that did not result in pregnancy were of greater mean diameter than embryos that resulted in pregnancy (P < 0.05). Pregnancy rates tended (P < 0.1) to be greater in recipients that were detected to be ovulating the same day or prior to that of the donor and that had been supplemented with progesterone (75 %) as opposed to untreated control mares of the same synchrony group (40 %). Progesterone supplementation did not affect the incidence of embryonic loss; however, there was a slightly higher loss of pregnancies between Day 15 and 30 in treated versus untreated recipients. There was no effect (P > 0.05) of treatment on pregnancy rate for embryos recovered from fertile versus subfertile donor mares. However, overall, there tended (P < 0.1) to be fewer pregnancies with embryos recovered from subfertile (50 %) as compared to fertile donors (75 %). It was concluded that supplemental progesterone at the dosage and frequency described was not beneficial in improving pregnancy rates in cyclic recipient mares after surgical embryo transfer.

Some observations on early embryonic death in mares

Early embryonic death (EED) was studied in 354 pregnant mares during two breeding seasons. Pregnant mares from six independent research projects were examined at 15, 20, 25, 30, 35, 40 and 50 days post-ovulation using a real-time ultrasound scanner. Mares that had previously been treated with anabolic steroids tended (P<0.10) toward a higher incidence of EED than untreated mares (21.1% vs 0%). Of the 59 pregnancies obtained from embryo transfer in 1982, 10 had undergone EED by day 50 (16.9%) compared to 10.1% for embryo transfer recipients in 1983. Overall, there was a 13.3% incidence of EED in embryo transfer recipients. This percentage was nearly identical to 13.4% incidence of EED for mares inseminated with fresh semen. In 1982, mares inseminated with frozen-thawed semen tended (P<0.10) to have a higher incidence of EED compared to those inseminated with fresh semen (34.6% vs 15.8%). However, in 1983 there was no difference (P>0.05) in the incidence of EED between mares inseminated with frozen-thawed and fresh semen (16.7% vs 12.5%). Further, combined over both years, there was no difference (P>0.10) in the incidence of EED (24.2% vs 14.4%). There was a 38.9% incidence of EED in infertile mares. Of the 354 pregnant mares, the overall incidence in EED through day 50 postovulation was 17.3%. The majority of EED (77.1%) occurred prior to day 35 post-ovulation. During the period 15 to 35 days post-ovulation, a greater (P<0.05) incidence of EED occurred between days 15 to 20 (26.2%) and 30 to 35 (29.5%) post-ovulation.

Control of cytathostome infections in mares treated at parturition with ivermectin

Six mares were treated on the day of parturition with an intramuscular injection of 0.2 mg kg −1 invermectin and placed in a pasture free of equine parasites as soon as possible after foaling. The mares and their foals were compared with a similar group of untreated mares and foals on an adjoining pasture. The experimental data was derived from mare and foal fecal egg counts, foal necropsies and pasture larval counts. Ivermectin administered to mares on the day of parturition, when combined with movement to parasite-free pastures, significantly lowered the cyathosome (small strongyle) egg production for 4 months. This reduced cyathostome exposure was reflected in lower worm-burdens in their foals for 5 months. The results indicate that ivermectin will effectively control equine strongyles when mares and their foals are moved to parasite-free pastures.

Estrogen-sensitive morphological plasticity inthe third ventricle of seasonally anovulatory mares.

A method was developed for fixation of equine brain tissue for examination by electron microscopy. Scanning electron microscopy (SEM) was used to examine the surface features of the third ventricle in seasonally anovulatory mares treated with estradiol-17β. The cellular population of the third ventricle of untreated mares was similar to that reported in other species: heavily ciliated cells covered the superior section of the third ventricle, ciliated cells became less dense inferiorly, and cells in the infundibular recess were well defined, covered with microvilli or miniblebs, and generally void of cilia. Supraependymal (SE) cells, which appeared to be type 1 as described in other species, were observed infrequently in the median eminence area of untreated mares. Treatment of seasonally anovulatory mares for 16 days with estradiol resulted in a decrease in the amount of ciliated cells and an increase in the number of SE cells in localized areas of the third ventricle. The apparent increase in supraependymal cell numbers in areas of the third ventricle other than the infundibular recess was observed in association with deciliation and therefore may be due to increased visibility of the cell surface in estradiol-treated mares. In the infundibular recess, increases in supraependymal cell numbers did not appear to be due to increased visibility of the cell surface since the amount of cilia in this area was not affected by estradiol treatment. These results suggest that estradiol has variable localized effects on the morphology of ependyma in the third ventricle of the horse not limited to the area of the median eminence but also affecting hypothalamic ependymal morphology.

Strongylus vulgaris infection in the horse.

The work described in this thesis is concerned with studies on various aspects of infection with one of the most common and pathogenic of the equine helminths, Strongylus vulgaris. In order to facilitate these experimental studies, an efficient technique was developed for the production of large numbers of S. vulgaris infective larvae in pure culture; in this, third stage S. vulgaris larvae were obtained by surgically transferring adult worms, collected from horses immediately after slaughter, directly into the caeca of worm-free yearling ponies. Subsequently, various investigations on the life-cycle, pathogenesis, epidemiology and immunity of S. vulgaris infection were carried out. Considerable controversy existed concerning the migratory route taken by developing parasitic larval stages of S. vulgaris in the tissues of the host. In order to elucidate this, worm-free pony foals were infected with a pure culture of infective larvae and killed at intervals over a period of 9 months. The results showed that infective larvae exsheathed and penetrated the intestine within a few days of infection. These larvae then moulted in the sub-mucosa, penetrated small arteries and had migrated within the lumina of the intestinal arteries to the anterior mesenteric site by 14 days after infection; in this predilection site larvae developed from early to late fourth stage. After a period of 3 - 4 months the fourth moult was completed and the young adults returned to the intestine again via the lumina of the arteries. Nodules were formed with the subsequent release of young adults into the intestinal lumen. The pre-patent period was 6-7 months. Studies on the pathogenesis of S. vulgaris infection showed that a distinct clinical syndrome occurred within 3-4 weeks of infection in foals given doses of 750 infective larvae. This syndrome was one of pyrexia, anorexia and colic associated with the presence of fourth stage larvae in the intestine and mesenteric arteries. Examination of tissues taken from foals killed over a period of 9 months demonstrated the sequence of pathological changes. During the course of these studies infected foals showed a marked polymorphonuclear leucocytosis and an eosinophilia together with an increase in the serum globulin levels; the latter appeared to be due mainly to increases in the p -globulin levels. Subsequently using 125I labelled albumin and 51Cr labelled red blood cells it was shown that the rate of albumin catabolism was increased and the red cell survival time reduced in ponies with patent infections. The epidemiology of equine helminthiasis was studied in 2 separate systems of management over 2 years. Basically 2 regimens were employed: one where untreated mares and foals went out in the spring to pastures never previously grazed by horses while in the other, mares, regularly treated with anthelmintics, and their foals grazed contaminated pasture. These systems allowed 2 sources of infection for susceptible foals: where mares were treated, the main source of infection was from overwintered larvae; where infected mares went out to clean pasture infection of the foals could only occur from larvae which developed from eggs passed in the faeces of the mare. The results demonstrated the following points. First, foals going out to grass in the spring can be infected by both overwintered larvae and by eggs passed in the faeces of their dams but the latter is by far the more important source of infection. Secondly, where horses remain untreated throughout the grazing season high levels of third stage strongyle larvae develop on pasture, whereas regular anthelmintic treatment at intervals of 2 - 4 weeks reduces pasture contamination to a minimum. Thirdly, although high levels of infective larvae may occur on pasture grazed by infected animals during the summer and autumn, these levels fall during the winter and by May the following year virtually no third stage larvae can be recovered from these pastures. The results of a series of experiments on the immunity to S. vulgaris infection provided the following Information. First, an age immunity was demonstrated when S-year-old worm-free ponies were infected with S. vulgaris larvae. No clinical signs or gross arterial lesions developed in these animals and no fourth stage larvae were recovered at necropsy. Secondly, a degree of acquired immunity in terms of resistance to reinfection was demonstrated in groups of naturally and experimentally infected animals. Finally, the results of a preliminary vaccination experiment have shown that the arterial lesions are less severe and that there is an 80% reduction in larval numbers in vaccinated foals compared to challenge controls.

48-HOUR ASSAY TEST FOR EQUINE GONADOTROPIN WITH RESULTS EXPRESSED IN INTERNATIONAL UNITS

OUR PREVIOUS METHODS for assaying equine gonadotropin (1, 2) involved autopsy of the test rats 120 hours after injection. The primary purpose for this long test interval was to obtain information on the qualitative ovarian response in follicular growth, luteinization, and interstitial cell development. This information was considered essential because, conceivably, the gonadotropic activity of pregnant mare serum might depend upon two or more gonadotropic hormones. We therefore wished to compare the nature of the responses obtained from sera collected at different intervals during pregnancy as well as the responses of various fractions obtained during studies on methods of purification. After comparing the qualitative and quantitative gonad response to extracts testing 4,000 to 7,000 R. U. per mg. total solids, with the response to untreated mare serum in normal and hypophysectomized male and female rats, we concluded that mare serum contains but one gonadotropic hormone (3). Three papers (4–7), give evid...

Response of Hypophysectomized Rats to Hihly Purified Extracts of Prenant Mare Serum

Goss and Cole1 have shown that extracts of mare serum can be prepared testing 4000 to 7000 R.U. per mg total solids (40,000 to 70,000 R.U. per mg nitrogen). The question arises regarding the nature of the hormone present in these extracts as compared to that in untreated mare serum. Evans, et al.,2 and Hellbaum3 have reported that more than one gonadotropic hormone is present in mare serum though these reports have not been confirmed. In the first mentioned paper the male rats showing only a Leydig tissue response were possibly sacrificed too soon to expect an effect upon the seminiferous epithelium. Further, had the time of autopsy of the females been delayed beyond 72 hours corpora lutea might have been encountered following the treatment with so-called FSH fractions. The extracts prepared by these authors were relatively crude preparations. We were interested, therefore, in determining whether or not highly purified materials would give similar evidence of fractionation of gonadotropic hormones. A frac...