E.M. Carnevale

Embryo technologies in the horse.

Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 degrees C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 microm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medicine, assisted reproductive techniques have been developed for the older, subfertile mare. Transfer of in vivo-matured oocytes from young, healthy mares into a recipients oviduct results in a 70-80% pregnancy rate compared with a 30-40% pregnancy rate when the oocytes are from older, subfertile mares. This procedure can also be used to evaluate in vitro maturation systems. In vitro production of embryos is still quite difficult in the horse. However, intracytoplasmic sperm injection (ICSI) has been used to produce several foals. Cleavage rates of 60% and blastocyst rates of 30% have been reported after ICSI of in vitro-matured oocytes. Gamete intrafallopian tube transfer (GIFT) is a possible treatment for subfertile stallions. Transfer of in vivo-matured oocytes with 200,000 sperm into the oviduct of normal mares resulted in a pregnancy rate of 55-82%. Oocyte freezing is a technique that has proven difficult in most species. However, equine oocytes vitrified in a solution of ethylene glycol, DMSO, and Ficoll and loaded onto a cryoloop resulted in three pregnancies of 26 transfers and two live foals produced. Production of a cloned horse appears to be likely, as several cloned pregnancies have recently been produced.

Relationships of age to uterine function and reproductive efficiency in mares

The uterine function and reproductive efficiency of 31 nonlactating pony mares were compared for two age groups: young (5 to 7 years, n=9) and old (>/=15 years, n=22). For pregnant mares, differences between age groups were not significant for the diameter of the largest follicle, cross-sectional area of the corpus luteum, growth profile of the embryonic vesicle or embryo mobility characteristics. Uterine contractility scores were lower (P<0.05), day of fixation of the embryonic vesicle was later (P<0.05), and uterine tone tended (P<0.10) to be lower in the old than the young mares. Endometrial biopsies in old mares had more (P<0.05) inflammatory cell infiltrations, more (P<0.05) fibrotic changes, and less dense (P<0.05) endometrial glands than in young mares. Ultrasonically detected intrauterine fluid collections were more extensive (P<0.05) in the old than the young mares. The pregnancy rate on Day 12 (Day 0=ovulation) was lower (P<0.05) and embryo-loss rate (Days 12 to 39) was greater (P<0.05) in old (32 and 62%, respectively) than in young (100 and 11%, respectively) mares. The results confirmed previous reports that old age was associated with increased endometrial inflammation, reduced pregnancy rate and increased embryo-loss rate. The results also indicated that uterine contractility and uterine tone were reduced and the fixation of the embryonic vesicle occurred later in old than in young mares.

Pregnancies from vitrified equine oocytes collected from super-stimulated and non-stimulated mares

The objectives were to compare embryo development rates after transfer into inseminated recipients, vitrified thawed oocytes collected from super-stimulated versus non-stimulated mares. In vivo matured oocytes were collected by transvaginal, ultrasound guided follicular aspiration from super-stimulated and non-stimulated mares 24-26 h after administration of hCG. Oocytes were cultured for 2-4 h prior to vitrification. Cryoprotectants were loaded in three steps before oocytes were placed onto a 0.5-0.7 mm diameter nylon cryoloop and plunged directly into liquid nitrogen. Oocytes were thawed and the cryoprotectant was removed in three steps. After thawing, oocytes were cultured 10-12 h before transfer into inseminated recipients. Non-vitrified oocytes, cultured 14-16 h before transfer, were used as controls. More oocytes were collected from 23 non-stimulated mares (20 of 29 follicles), than 10 super-stimulated mares (18 of 88 follicles; P < 0.001). Of the 20 oocytes collected from non-stimulated mares, 12 were vitrified and 8 were transferred as controls. After thawing, 10 of the 12 oocytes were morphologically intact and transferred into recipients resulting in one embryonic vesicle on Day 16 (1 of 12 = 8%). Fourteen oocytes from super-stimulated mares were vitrified, and 4 were transferred as controls. After thawing, 9 of the 14 oocytes were morphologically intact and transferred into recipients resulting in two embryonic vesicles on Day 16 (2 of 14 = 14%). In control transfers, 7 of 8 oocytes from non-stimulated mares and 3 of 4 oocytes from super-stimulated mares resulted in embryonic vesicles on Day 16. The two pregnancies from vitrified oocytes resulted in healthy foals.

Embryo development rates after transfer of oocytes matured in vivo, in vitro, or within oviducts of mares

Objectives of the present study were to use oocyte transfer: 1) to compare the developmental ability of oocytes collected from ovaries of live mares with those collected from slaughterhouse ovaries; and 2) to compare the viability of oocytes matured in vivo, in vitro, or within the oviduct. Oocytes were collected by transvaginal, ultrasound-guided follicular aspiration (TVA) from live mares or from slicing slaughterhouse ovaries. Four groups of oocytes were transferred into the oviducts of recipients that were inseminated: 1) oocytes matured in vivo and collected by TVA from preovulatory follicles of estrous mares 32 to 36 h after administration of hCG; 2) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and matured in vitro for 36 to 38 h; 3) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and transferred into a recipients oviduct <1 h after collection; and 4) im mature oocytes collected from slaughterhouse ovaries containing a corpus luteum and matured in vitro for 36 to 38 hours. Embryo development rates were higher (P < 0.001) for oocytes matured in vivo (82%) than for oocytes matured in vitro (9%) or within the oviduct (0%). However, neither the method of maturation nor the source of oocytes affected (P > 0.1) embryo development rates after the transfer of immature oocytes.

Aging effects on follicular activity and concentrations of FSH, LH, and progesterone in mares

Abstract Follicular activity and daily circulating concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), and progesterone were compared between young and old mares. Mares were divided into the following age groups: Group 1, 5–7 years ( n =9); Group 2, 15–19 years ( n =11); Group 3, 20 or more years ( n =11). The experiment encompassed three consecutive ovulatory periods for each mare; the interovulatory interval after the second ovulation was shortened by the administration of prostaglandin F 2α (PGF 2α ). Follicular activity was assessed by daily transrectal ultrasonic imaging from 5 days prior to the first ovulation to 3 days after the second ovulation. The diameters of the largest and second-largest follicles and the numbers of follicles in different diameter categories (3–5, 6–10, 11–15, 16–20, and 20 or more mm) were recorded. On Day 8 after the second ovulation, 5 mg of PGF 2α were administered, and ultrasonic imaging was used to detect the day of the third ovulation. Day of ovulation was defined as Day 0. Significant effects of age on follicular activity included a group effect ( P P P P P P P P P 2α treatment to ovulation than mares in Groups 1 and 2. These findings corresponded with a smaller ( P P P

Follicular activity and concentrations of FSH and LH associated with senescence in mares

The nature of reproductive senescence was studied during the expected ovulatory season in pony mares by transrectal ultrasonic monitoring of the ovaries and assay of circulating concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH). Old mares (⩾20 years) were less likely (P<0.005) to ovulate at regular intervals than younger mares as indicated by the number of mares with two consecutive interovulatory intervals (IOI; 5–7 years, 9/9; 15–19 years, 10/10; ⩾20 years, 8/16). In addition, a central area of nonechogenic fluid (unknown source) was detected at the ovulatory site in more (P<0.005) ovulations in old mares than in younger mares (5–7 years, 1/91; 15–19 years, 4/78; ⩾20 years, 10/68). Old mares were grouped as follows: (1) normal length of the follicular phase (interval from luteolysis to ovulation) of the IOI (n=9); (2) elongated follicular phase (>2 SD from the mean of all mares with an IOI) (n=2); (3) one ovulation in 80 days (n=2); and (4) no ovulations or follicles ⩾5 mm in 60 days (n=3). Mares with an IOI but with an elongated, compared with a normal length of the follicular phase, had fewer follicles per ovulatory wave (1.5 ± 0.5, 3.1 ± 0.3, P< 0.05), a longer IOI (30.0 ± 1.0, 25.8 ± 0.6 days, P < 0.01), a longer interval from administration of a luteolytic dose of PGF2α to ovulation (29.5 ±8.5, 15.8 ± 0.9 days, P < 0.02), and higher mean concentrations of FSH (13.0 ± 3.4, 3.3 ± 0.6 ng ml−1, P < 0.001) and LH (18.5 ± 5.3, 7.6 ± 1.5 ng ml−1, P < 0.02) during the follicular phase. Mean concentrations of FSH, LH and progesterone during the luteal phase were not different among ovulating mares; however, during the follicular phase, concentrations of FSH and LH were significantly different among groups with the lowest mean concentrations occurring in mares with a follicular phase of normal length. Mean concentrations of FSH for mares that did not ovulate were higher (P<0.05) than for the 3 days encompassing ovulation in the mares with an IOI and tended to be higher (P<0.08) than during the 3 days prior to luteolysis (period of maximal FSH concentrations). Results indicated that ovarian senescence in these mares involved the following progression: lengthening of the follicular phase, sporadically occurring ovulations, and a final state of persistent follicular inactivity (follicles ≤5 mm). Ovarian senescence was accompanied by elevated circulating concentrations of gonadotropins.

Use of a linear ultrasonic transducer for the transvaginal aspiration and transfer of oocytes in the mare

Summary A 5 MHz linear ultrasonic transducer was used for the transvaginal aspiration and transfer of oocytes through the use of an ultrasonically-guided needle. Mares (n=18) with a follicle ≤30 mm were given human chorionic gonadotropin (hCG); follicular aspirations were attempted between 17 to 28 hours (mean of 24 hours) later. Oocytes were recovered from 14 of 18 aspirations (78% collection rate), and intrafollicular oocyte transfers were attempted in 7 recipients. Recipients were given an injection of hCG approximately 24 hours prior to transfer of an oocyte into the follicle. Two of the 7 oocyte transfers resulted in twin conceptuses. The techniques used in this preliminary study resulted in the recovery of equine follicular oocytes and in the successful intrafollicular transfer of oocytes.

Heterogenous and xenogenous fertilization of in vivo matured equine oocytes

Summary Forty-five in vivo matured equine oocytes were recovered from 63 follicular aspiration attempts (71.4%). HCG did not improve recovery rate (65% — 24/37 for treated vs 81% — 21/26 for nontreated mares). Fifteen oocytes were transferred into the oviduct of inseminated recipient mares (heterogenous fertilization) and 15 oocytes plus equine spermatozoa were transferred into rabbit oviducts (xenogenous fertilization). Ten oocytes (3 fertilized) were recovered from recipient mare oviducts following removal and flushing two days after transfer. Eight oocytes (nonfertilized) were recovered from rabbit oviducts. Oviductal transfer into separate recipient mares of three embryos produced from heterogenous fertilization resulted in two pregnancies. One mare produced a normal live foal and the other mare aborted at 20 days of gestation. Results from these studies suggest that: 1) a reliable method for collection of in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from valuable infertile mares that fail to get pregnant or produce embryos by standard methods.

Strategies to improve the ovarian response to equine pituitary extract in cyclic mares

Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation; (3) administration of EPE twice daily in successively decreasing doses. In the first experiment, 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily; (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of >35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation, or two doses of a GnRH-a agonist upon detection of a follicle >35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles >35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF2, and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF2alpha, but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles > or = 35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose, improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates.

Age and pasture effects on vernal transition in mares

The objectives of the present study were to determine if follicular activity was less in old than in young mares during the spring transition and if green pasture would hasten onset of the ovulatory season. Experiments were conducted over 2 sequential years using young mares (3 to 7 yr) and old mares (> or =14 yr). In Experiment 1, growth of the largest and second-largest follicles were compared for young mares (5 to 7 yr) and old mares (> or =14 yr) for 21 d prior to the first ovulation of the year. More follicular activity was noted in young than in old mares. Main effect of age was significant for diameter of the largest follicle, and interaction of day-by-age was significant for diameter of the second-largest follicle. Prior to the beginning of the breeding season, the mares were randomly divided into dry-lot and pasture groups. The interval from May 2 to ovulation was shorter (P < 0.005) for mares put on pasture on May 2 than for mares kept in dry lot (means +/- SEM, 14.5 +/- 2.7 and 21.3 +/- 3.2 d, respectively). In Experiment 2, follicular activity was compared among 3 age groups (3 to 7, 17 to 19, and > or =20 yr). The total number of follicles > or =10 mm was higher (P < 0.05) for young mares and lower (P < 0.05) for old mares than for mares of an intermediate age. Main effect of age and interaction of day-by-age were significant for diameter of largest and second-largest follicles, being smaller for mares > or =20 yr than for younger mares. The interval from development of a follicle > or =30 mm to ovulation was shorter (P < 0.05) for mares placed on pasture when a > or =30 mm follicle developed than the interval for mares kept in dry lot (5.7 +/- 0.7 and 8.2 +/- 0.9 d, respectively). In summary, less follicular activity occurred in old than in young mares during the transitional period, and mares pastured on green grass ovulated sooner in the spring than mares housed on dry lot and fed hay.