Preliminary study of the contraceptive effect of a self-assembling intrauterine device (iUPODs) in mares maintained in a paddock with a fertile stallion.

There is an urgent need for practical methods of population control (i.e., contraception and/or sterilization) for free-roaming (i.e., "wild" or "feral") horses and burros on Western Public Lands in the United States. The objective of this study was to evaluate the contraceptive efficacy of a novel self-assembling three-part polymer-coated magnetic intrauterine device termed as an intrauterine POD (self-assembling; iUPOD) when there are natural breeding conditions when iUPOD use was managed by veterinary professionals with no prior experience with the device. Six mares were administered an iUPOD and were then housed continuously with a fertile stallion for 91 days. The intrauterine POD retention and contraceptive efficacy were 100%. Two mares had prolonged corpus luteum function (for 37 and 91 days) immediately after iUPOD placement. For the estrous cycles of the other mares, the duration of diestrus was 7.8 ± 2.7 days (mean ± S.D.). Four of the mares (67%) became pregnant when in a paddock with the same stallion the year after iUPOD removal. These results are encouraging for use of the iUPOD as a practical and reversible method of fertility control in free-roaming horses and burros.

Effect of Chronic Administration of Oxytocin on Corpus Luteum Function in Cycling Mares.

The objective of this study was to determine if intramuscular administration of 60 units of oxytocin once daily for 29 days, regardless of when treatment was initiated during the estrous cycle (i.e., without monitoring estrous behavior and/or detecting ovulation), would induce prolonged corpus luteum (CL) function in cycling mares. Mares were randomly assigned to two groups: (1) saline-treated control (n = 7) and (2) oxytocin-treated (n = 9) subjects. Control mares received 3 cc of saline, and oxytocin-treated mares received 60 units (3 cc) of oxytocin intramuscularly for 29 consecutive days. Treatment was initiated in all mares on the same day (day 1), independent of the day of the cycle. Jugular blood samples for determination of progesterone concentration were collected three times weekly (M, W, and F) for 21 days before treatment was initiated to confirm that all mares had a luteal phase of normal duration immediately before treatment. Beginning on the first day of treatment, blood samples were collected daily for eight days and then three times weekly through day 80. Mares were considered to have prolonged CL function if serum progesterone remained >1.0 ng/mL continuously for at least 25 days after the end of the treatment period. The proportion of mares with prolonged CL function was higher in the oxytocin-treated group than in the saline-treated group (7/9 vs. 1/7, respectively; P < .05). Three of the seven oxytocin-treated mares that developed prolonged CL function initially underwent luteolysis within 4-7 days of the start of oxytocin treatment and then developed prolonged CL function after the subsequent ovulation during the treatment period. In the other four oxytocin-treated mares that developed prolonged CL function, progesterone remained >1.0 ng/mL throughout the treatment period and into the post-treatment period. All mares with prolonged CL function maintained elevated progesterone concentrations through at least day 55 of the study. In conclusion, intramuscular administration of 60 units of oxytocin for 29 consecutive days effectively prolonged CL function in mares, regardless of when treatment was initiated during the estrous cycle. Importantly, this represents a protocol for using oxytocin treatment to prolong CL function that does not require detection of estrous behavior or day of ovulation.

Evaluation of a Proprietary Slow-Release Oxytocin Formulation on Corpus Luteum Function in Mares.

Prolonging function of the corpus luteum (CL) is a method of suppressing estrus that relies on continued secretion of endogenous progesterone to keep mares out of heat naturally. The use of oxytocin treatment to prolong CL function is gaining increasing use, and the most common treatment protocol involves administration of 60 units of oxytocin intramuscularly (IM) once daily on days 7-14 after ovulation (eight daily treatments). Although that protocol induces prolonged CL function in ≥70% of treated mares, the need for daily administration is a drawback to its use. Therefore, the objective of this study was to evaluate the efficacy of a proprietary slow-release oxytocin formulation (SR-OT) for prolonging CL function that requires only two treatments. Mares were examined via transrectal palpation and ultrasonography to determine the day of ovulation (day 0) and then randomly assigned to a nontreated control group and an SR-OT treatment group (n = 8 mares/group). Mares in the treated group received 1.0 mL of SR-OT containing 2,400 IU oxytocin IM once on day 7 and again on day 10 after ovulation. Jugular blood samples were collected on day 0 and then every Monday, Wednesday, and Friday for 50 days for determination of the serum progesterone concentration. Mares were classified as having prolonged CL function if their progesterone concentration remained >1.0 ng/mL continuously for at least 30 days. Corpus luteum function was prolonged in 0/8 (0%) control mares and 6/8 (75%) of the SR-OT-treated mares (P < .01). The demonstrated efficacy of this two-injection, SR-OT protocol represents a 75% reduction in the number of oxytocin treatments compared with daily administration of oxytocin from day 7-14, making it a more practical treatment protocol.

Expression profiles of select genes in cumulus-oocyte complexes from young and aged mares.

There is compelling evidence that oocytes from mares >18 years of age have a high incidence of inherent defects that result in early embryonic loss. In women, an age-related decrease in oocyte quality is associated with an increased incidence of aneuploidy and it has recently been determined that the gene expression profile of human oocytes is altered with advancing age. We hypothesised that similar age-related aberrations in gene expression occur in equine oocytes. Therefore, the aim of the present study was to compare gene expression profiles of individual oocytes and cumulus cells from young and aged mares, specifically evaluating genes that have been identified as being differentially expressed with advancing maternal age and/or aneuploidy in human oocytes. Expression of 48 genes was compared between 14 cumulus-oocyte complexes (COCs) from mares aged 3-12 years and 10 COCs from mares ≥18 years of age. Three genes (mitochondrial translational initiation factor 3 (IF3), heat shock transcription factor 5 (HSF5) and Y box binding protein 2 (YBX2)) were differentially expressed in oocytes, with all being more abundant in oocytes from young mares. Three genes (ADP-ribosylation factor-like 6 interacting protein 6 (ARL6IP6), BCL2-associated X protein (BAX) and hypoxia upregulated 1 (HYOU1)) were differentially expressed in cumulus cells, with all being more abundant in aged mares. The results of the present study confirm there are age-related differences in gene expression in equine COCs, which may be associated with the lower quality and decreased developmental competence of oocytes from aged mares.

Parental genomes mix in mule and human cell nuclei.

Whether chromosome sets inherited from father and mother occupy separate spaces in the cell nucleus is a question first asked over 110 years ago. Recently, the nuclear organization of the genome has come increasingly into focus as an important level of epigenetic regulation. In this context, it is indispensable to know whether or not parental genomes are spatially separated. Genome separation had been demonstrated for plant hybrids and for the early mammalian embryo. Conclusive studies for somatic mammalian cell nuclei are lacking because homologous chromosomes from the two parents cannot be distinguished within a species. We circumvented this problem by investigating the three-dimensional distribution of chromosomes in mule lymphocytes and fibroblasts. Genomic DNA of horse and donkey was used as probes in fluorescence in situ hybridization under conditions where only tandem repetitive sequences were detected. We thus could determine the distribution of maternal and paternal chromosome sets in structurally preserved interphase nuclei for the first time. In addition, we investigated the distribution of several pairs of chromosomes in human bilobed granulocytes. Qualitative and quantitative image evaluation did not reveal any evidence for the separation of parental genomes. On the contrary, we observed mixing of maternal and paternal chromosome sets.

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.

Equine cloning: applications and outcomes.

Cloning is one of several new assisted reproductive techniques being developed for clinical use in the equine industry. Potential uses of equine cloning include: (1) the preservation of genetics from individual animals that would otherwise not be able to reproduce, such as geldings; (2) the preservation of genetic material of endangered and/or exotic species, such as the Mongolian wild horse (Przewalski's horse); and (3) because of the companion animal role that horses fill for some individuals, it is likely that some horse owners will have individual animals cloned for emotional fulfillment. Although equine cloning has been successful, like other species, it remains a very inefficient process (<3% success). In most species, the inefficiency of cloning results from a high incidence of embryonic, fetal and/or placental developmental abnormalities that contribute to extremely high rates of embryonic loss, abortion and stillbirths throughout gestation and compromised neonatal health after birth. The present review describes some of the ultrasonographic, endocrinological and histopathological characteristics of successful (produced viable offspring) and unsuccessful (resulted in pregnancy failure) cloned equine (mule and horse) pregnancies we have produced. A total of 21 cloned mule pregnancies were established using fetal fibroblast cells, whereas a total of seven cloned horse pregnancies were established using adult cumulus cells. Three of the cloned mule conceptuses were carried to term, resulting in the birth of three healthy clones. This information adds to an accumulating body of knowledge about the outcome of cloned equine pregnancies, which will help to establish when, and perhaps why, many cloned equine pregnancies fail.

Effect of repeated transvaginal ultrasound-guided follicle aspiration on fertility in mares.

OBJECTIVE: To determine whether performance of transvaginal ultrasound-guided follicle aspiration (TVUFA) repeatedly in mares adversely affects their fertility. DESIGN: Historical prospective study. ANIMALS: 23 mares that had never undergone TVUFA and 59 mares that had undergone TVUFA on 1 to 11 occasions. PROCEDURE: Mares were classified into 4 groups according to the number of TVUFA procedures previously performed on the ovary in which ovulation occurred at the time of insemination as follows: group 1, 0 TVUFAs (control group, n = 23 mares); group 2, 1 or 2 TVUFAs (40 mare-cycles); group 3, 3 or 4 TVUFAs (21 mare-cycles); and group 4, 5 to 11 TVUFAs (13 mare-cycles). Each ovary and its associated number of TVUFAs were considered separately; therefore, some of the mares that underwent TVUFA were represented in > 1 group (1 mare was included in group 2 twice [once for each ovary]), and the sample size in groups 2, 3, and 4 was denoted as mare-cycles. Fertility was assessed as pregnancy rates in cycles in which mares were inseminated with fresh or cooled semen from 1 fertile stallion. RESULTS: There were no significant differences in pregnancy rates among groups 1, 2, 3, and 4 (83%, 90%, 81%, and 85%, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that repeated performance of TVUFA (as many as 11 times) had no detectable adverse effect on fertility in mares. This finding is clinically important for situations when TVUFA is performed on fertile mares, whether for oocyte collection or other purposes.

Effect of homologous follicular fluid from medium-sized and large follicles on in vitro maturation of equine cumulus-oocyte complexes.

The in vitro maturation (IVM) of equine oocytes is typically performed using various synthetic media; however, an optimal IVM system for equine oocytes has not been developed. The aim of the present study was to evaluate the effects of two types of follicular fluid (FF) obtained from cyclic mares and two incubation intervals for the IVM of equine cumulus-oocyte complexes (COCs). Follicular fluid was collected from medium-sized (20-29 mm diameter) and large (e30 mm; post-human chorionic gonadotrophin administration) follicles using transvaginal ultrasound-guided follicle aspiration. Compact (n = 232) and non-compact (n = 183) COCs obtained from a slaughterhouse were incubated separately in the following groups: (1) FF from medium follicles for 24 h; (2) FF from large follicles for 24 h; (3) control (synthetic) medium for 24 h; (4) FF from medium follicles for 24 h then FF from large follicles for an additional 24 h; (5) FF from large follicles for 48 h; and (6) control medium for 48 h. For compact COCs, there was a tendency (P = 0.06) for more COCs incubated in FF from large follicles for 24 h to reach metaphase II compared with those incubated in control medium for 24 h (58% v. 35%, respectively). More (P < 0.05) compact COCs had degenerated after incubation in control medium for 48 h compared with all other groups (51% v. 14-24%, respectively). For non-compact COCs, incubation in FF from medium follicles for 24 h resulted in more (P = 0.05) COCs at metaphase II compared with control medium for 48 h (58% v. 29%, respectively). These results indicate that homologous FF from cyclic mares is a suitable alternative for the IVM of equine COCs and that it may be superior to conventional media for longer (i.e. >24 h) incubation intervals.

Cloned horse pregnancies produced using adult cumulus cells.

The objectives of the present study were to: (1) clone horses using adult cumulus cells; and (2) determine whether the cumulus cell donor affected the outcome. In vivo-matured cumulus-oocyte complexes were obtained using transvaginal ultrasound-guided follicle aspiration; oocytes were used as cytoplasts, whereas cumulus cells (from one of three different mares) were used as donor cells. Immediately following nuclear transfer and activation procedures, cloned embryos were transferred surgically to the oviduct of recipient mares (n = 2-5 embryos per recipient) that had ovulated within 24 h prior to the transfer. An initial pregnancy examination was performed between Days 14 and 16 (Day 0 = surgery); subsequent examinations were then performed every 7-10 days. A total of 136 follicles were aspirated in 96 mares, from which 72 oocytes were recovered (53%). Sixty-two cloned embryos were transferred to recipient mares, which resulted in seven (11.3%) ultrasonographically detectable conceptuses between Days 14 and 16. All seven conceptuses were lost spontaneously between Days 16 and 80. Cumulus cells from Mare 160 tended (P = 0.08) to result in a higher embryo survival rate than cumulus cells from Mare 221 (4/17 v. 1/25 respectively). To our knowledge, this is the first report documenting the establishment of cloned equine pregnancies derived from adult cumulus cells.

Effect on fertility of uterine lavage performed immediately prior to insemination in mares.

OBJECTIVE: To determine the effect on fertility of large-volume uterine lavage with lactated Ringer's solution (LRS) performed immediately prior to insemination in mares. DESIGN: Prospective randomized controlled study. ANIMALS: 20 mares. PROCEDURE: Control mares (n = 10) were inseminated with 1 billion (estimated before cooling) progressively motile spermatozoa that had been cooled in a passive cooling unit for 24 hours. Mares (n = 10) in the treatment group were inseminated with 1 billion progressively motile spermatozoa (cooled as described for control mares) immediately after uterine lavage with 4 L of sterile LRS. RESULTS: There were no significant differences in pregnancy rates or size of the embryonic vesicle on days 12, 13, and 14 after ovulation between control and treated mares. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that uterine lavage with LRS can be performed immediately prior to insemination without adversely affecting fertility in mares. This is clinically important, because insemination may be necessary when a mare has inflammation-associated fluid (detectable ultrasonographically) in the uterus; removal of the fluid is desirable, because it adversely affects spermatozoal motility and fertility. This situation typically arises when mares require rebreeding after they have developed persistent mating-induced endometritis or are inseminated multiple times in a 24-hour period (during the period of physiologic mating-induced inflammation), which is a common practice when using cooled or frozen-thawed semen.