A simplified grading system for in vivo and in vitro derived vitrified equine embryos.

The ability to predict the viability of embryos before vitrification and thawing has important commercial applications in any breeding program. The aim of this study was to develop and evaluate a simplified embryo grading system for both in vivo- and in vitro-derived vitrified day 8 embryos. The in vivo derived (n=109) and in vitro - intracytoplasmic sperm injection derived (n=145) embryos were produced in a commercial embryo program. The embryos were classified as Grade 1, 2 or 3 based on the amount of extruded material between the trophoblast and the zona pellucida observed during the vitrification process. The embryos were vitrified at day 8 of development in a two-step system with increasing concentrations of dimethylsulfoxide and ethylene glycol and 0.5 M sucrose in the final solution. Each embryo was thawed in 0.3 M and then 0.15 M sucrose before transfer into holding medium for non-surgical transfer into a recipient mare. Analysis of the relationship between the embryo grading system and pregnancy rates after vitrification, thawing and transfer of in vivo and in vitro derived embryos confirmed that there was a significant effect of origin (in vivo vs in vitro; P ≤ 0.05), and embryo grade (P ≤ 0.001) on embryo survival after transfer. In conclusion, this simplified grading system is predictive of embryo survival for both in vivo- and in vitro- derived vitrified equine embryos.

Equine maternal aging affects the metabolomic profile of oocytes and follicular cells during different maturation time points.

Introduction: Oocyte quality and fertility decline with advanced maternal age. During maturation within the ovarian follicle, the oocyte relies on the associated somatic cells, specifically cumulus and granulosa cells, to acquire essential components for developmental capacity. Methods: A nontargeted metabolomics approach was used to investigate the effects of mare age on different cell types within the dominant, follicular-phase follicle at three time points during maturation. Metabolomic analyses from single oocytes and associated cumulus and granulosa cells allowed correlations of metabolite abundance among cell types. Results and Discussion: Overall, many of the age-related changes in metabolite abundance point to Impaired mitochondrial metabolic function and oxidative stress in oocytes and follicular cells. Supporting findings include a higher abundance of glutamic acid and triglycerides and lower abundance of ceramides in oocytes and somatic follicular cells from old than young mares. Lower abundance of alanine in all follicular cell types from old mares, suggests limited anaerobic energy metabolism. The results also indicate impaired transfer of carbohydrate and free fatty acid substrates from cumulus cells to the oocytes of old mares, potentially related to disruption of transzonal projections between the cell types. The identification of age-associated alterations in the abundance of specific metabolites and their correlations among cells contribute to our understanding of follicular dysfunction with maternal aging.

Maturation and culture affect the metabolomic profile of oocytes and follicular cells in young and old mares.

Introduction: Oocytes and follicular somatic cells within the ovarian follicle are altered during maturation and after exposure to culture in vitro. In the present study, we used a nontargeted metabolomics approach to assess changes in oocytes, cumulus cells, and granulosa cells from dominant, follicular-phase follicles in young and old mares. Methods: Samples were collected at three stages associated with oocyte maturation: (1) GV, germinal vesicle stage, prior to the induction of follicle/oocyte maturation in vivo; (2) MI, metaphase I, maturing, collected 24 h after induction of maturation in vivo; and (3) MIIC, metaphase II, mature with collection 24 h after induction of maturation in vivo plus 18 h of culture in vitro. Samples were analyzed using gas and liquid chromatography coupled to mass spectrometry only when all three stages of a specific cell type were obtained from the same mare. Results and Discussion: Significant differences in metabolite abundance were most often associated with MIIC, with some of the differences appearing to be linked to the final stage of maturation and others to exposure to culture medium. While differences occurred for many metabolite groups, some of the most notable were detected for energy and lipid metabolism and amino acid abundance. The study demonstrated that metabolomics has potential to aid in optimizing culture methods and evaluating cell culture additives to support differences in COCs associated with maternal factors.

Tesserae on Venus may preserve evidence of fluvial erosion.

Fluvial erosion is usually assumed to be absent on Venus, precluded by a high surface temperature of ~450 °C and supported by extensive uneroded volcanic flows. However, recent global circulation models suggest the possibility of Earth-like climatic conditions on Venus for much of its earlier history, prior to catastrophic runaway greenhouse warming. We observe that the stratigraphically oldest, geologically most complex units, tesserae, exhibit valley patterns morphologically similar to the patterns resulting from fluvial erosion on Earth. Given poor topographic resolution, we use an indirect technique to recognize valleys, based on the pattern of lava flooding of tesserae margins by adjacent plains volcanism. These observed valley patterns are attributed to primary geology, tectonic deformation, followed by fluvial erosion (and lesser wind erosion). This proposed fluvial erosion in tesserae provides support for climate models for a cool, wet climate on early Venus and could be an attractive research theme for future Venus missions.

The effect of appropriate bladder management on urinary tract infection rate in patients with a new spinal cord injury: a prospective observational study.

PURPOSE: This study aimed to determine the rate of urinary tract infection (UTI) in patients with a new spinal cord injury (SCI) and identify which bladder management technique is associated with the lowest rate of UTI. METHODS: Adults admitted to the Victorian Spinal Cord Service with a new SCI from 2012 to 2014 were enrolled. Data collected included patient characteristics, SCI level, bladder management and diagnosis of UTI. Bacteriuria (≥ 102 colony-forming organisms/mL) with clinical signs of infection was used to define a UTI. RESULTS: 143 patients were enrolled. 36 (25%) were female; the median age was 42 years. An indwelling urethral catheter (IUC) was placed in all the patients initially. 55 (38%) patients developed a UTI with an IUC, representing a UTI rate of 8.7/1000 inpatient days. Long-term bladder management strategies were initiated after a median of 58 days. IUC removal and initiation of any other alternative bladder management halved the UTI rate to 4.4/1000 inpatient days, p < 0.001. Intermittent self-catheterisation (ISC) and suprapubic catheter placement had lower UTI rates compared to IUC, 6.84 and 3.81 UTI/1000 inpatient days, p = 0.36 and p = 0.007, respectively. An IUC was re-inserted in 29 patients and resulted in a higher UTI rate of 8.33/1000 inpatient days. CONCLUSION: This study has identified a high UTI rate in new SCI patients with an IUC and reinforces the importance of early IUC removal and initiation of non-IUC bladder management in this cohort of patients.

CD23/FcεRII: molecular multi-tasking.

CD23 is the low-affinity receptor for immunoglobulin (Ig)E and plays important roles in the regulation of IgE responses. CD23 can be cleaved from cell surfaces to yield a range of soluble CD23 (sCD23) proteins that have pleiotropic cytokine-like activities. The regions of CD23 responsible for interaction with many of its known ligands, including IgE, CD21, major histocompatibility complex (MHC) class II and integrins, have been identified and help to explain the structure-function relationships within the CD23 protein. Translational studies of CD23 underline its credibility as a target for therapeutic intervention strategies and illustrate its involvement in mediating therapeutic effects of antibodies directed at other targets.

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros.

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n=9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.

Methanol as a cryoprotectant for equine embryos.

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.

In vitro maturation and transfer of equine oocytes after transport of ovaries at 12 or 22 degrees C.

Transportation of equine ovaries would allow shipment of oocytes for research purposes or transfer after the death of a valuable mare. The objective of this study was to compare two temperatures for maintaining ovaries during a transport interval of 18-24 h. The goal was to obtain pregnancies after transport of ovaries, maturation of oocytes in vitro, and transfer of oocytes. Each shipment was composed of ovaries four to seven mares collected from an abattoir. From each mare, one ovary was packaged at approximately 12 degrees C, and the other was packaged at approximately 22 degrees C. Upon arrival at our laboratory, oocytes were collected and cultured for 24 h. For each transfer, between 9 and 15 oocytes from each group were placed into the oviducts of estrous mares through standing flank laparotomies. Recipients received human chorionic gonadotropin (hCG; 2000 IU, i.v.) 30-36 h before transfer (to synchronize ovulation). Recipients were inseminated 18-20 h before transfers with 2 x 10(9) progressively motile sperm. Uteri of recipients were examined with ultrasound to determine the number of developing embryos. On Day 16 ( ovulation = day 0), developing embryos were recovered by uterine lavage. Parentage verification was performed on recovered vesicles. Pregnancy rates were analyzed by Chi-square. The percentage of oocytes that developed into embryonic vesicles on Day 16 was not different between transport temperatures (22 degrees C, 13/73, 18% versus 12 degrees C, 11/73, 15%). In conclusion, pregnancies were obtained from in vitro matured oocytes that were recovered from ovaries transported for 18-24h at 12 or 22 degrees C.

Oocyte transfer in mares with intrauterine or intraoviductal insemination using fresh, cooled, and frozen stallion semen.

The objectives were to compare embryo development rates after oocyte transfer with: (1) intrauterine or intraoviductal inseminations of fresh semen versus intraoviductal insemination of frozen semen; (2) intraoviductal versus intrauterine inseminations of cooled semen. In Experiment I, oocytes were transferred into the oviduct, and recipients were inseminated into the uterus with 1 x 10(9) fresh spermatozoa, or into the oviduct with 2 x 10(5) fresh or frozen-thawed spermatozoa. In Experiment II, semen was cooled to 5 degrees C before intrauterine insemination with 2 x 10(9) spermatozoa or intraoviductal inseminations of 2 x 10(5) spermatozoa (deposited with the oocytes). In Experiment I, embryo development rates were similar (P>0.05) for intrauterine versus intraoviductal inseminations when fresh semen was used (8/14, 57% and 9/11, 82%, respectively). However, embryo development rates were lower (P<0.05) when frozen spermatozoa were placed within the oviduct (1/12, 8%). In Experiment II, embryo development rates were higher (P<0.05) when cooled semen was used for intrauterine (19/23, 83%) versus intraoviductal (4/16, 25%) inseminations. We concluded that intraoviductal insemination can be successfully performed using fresh spermatozoa. However, the use of cooled and frozen spermatozoa for intraoviductal inseminations was less successful, and needs further investigation.

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.

Effect of time of oocyte collection and site of insemination on oocyte transfer in mares.

The objective of the study was to compare embryo development rates after transfer of oocytes collected 22 or 33 h after hCG injection into recipients inseminated within the uterus or the oviduct. Oocytes were collected at approximately 22 or 33 h after hCG injections and incubated for approximately 16 or 1.5 h, respectively, before transfer. Intrauterine inseminations using 1 x 10(9) progressively motile sperm were done approximately 12 h before and 2 h after transfer. For intraoviductal inseminations (gamete intrafallopian transfer [GIFT]), semen was centrifuged through a Percoll gradient, and 200,000 progressively motile sperm were transferred with oocytes into the oviduct. Time of oocyte collection (22 or 33 h) after hCG injection did not affect embryo development rates (17/25, 68%, vs 12/23, 52%, respectively; P = 0.40). When results from oocyte collections at 22 and 33 h after hCG were combined, oocyte transfer with intraoviductal vs intrauterine insemination resulted in similar (P = 0.70) embryo development rates (12/22, 55%, and 17/26, 65%, respectively). However, the interaction between time of oocyte collection and site of insemination tended to be significant (P = 0.09), suggesting that GIFT using oocytes collected at 33 h after hCG may not be as effective as using oocytes collected at 22 h after hCG. Because intraoviductal insemination requires a low number of sperm, GIFT could be used in cases of male subfertility, frozen semen, or sexed sperm.

Equine sperm-oocyte interaction: results after intraoviductal and intrauterine inseminations of recipients for oocyte transfer.

Insemination of recipients for oocyte transfer and gamete intrafallopian transfer (GIFT) in five experiments were reviewed, and factors that affected pregnancy rates were ascertained. Oocytes were transferred into recipients that were (1) cyclic and ovulated at the approximate time of oocyte transfer, (2) cyclic with aspiration of the preovulatory follicle, and (3) noncyclic and treated with hormones. Recipients were inseminated before, after, or before and after transfer. Intrauterine and intraoviductal inseminations were done. Pregnancy rates were not different between cyclic and noncyclic recipients (8/15, 53% and 37/93, 39%). The highest numerical pregnancy rates resulted when recipients were inseminated with fresh semen from fertile stallions before oocyte transfer or inseminated with cooled transported semen before and after oocyte transfer. Oxytocin was administered to recipients before oocyte transfer when fluid was imaged within the uterus. Administration of oxytocin to recipients at the time of oocyte transfer resulted in significantly higher pregnancy rates than when oxytocin was not administered (17/26, 65% and 28/86, 33%). Intraoviductal and intrauterine inseminations of recipients during oocyte transfer resulted in similar embryo development rates when fresh semen was used (12/22, 55% and 14/26, 55%). However, embryo development rates significantly reduced when frozen (1/21, 5%) versus fresh sperm were inseminated into the oviduct. Results suggest that insemination of a recipient before and after transfer could be beneficial when semen quality is not optimal; however, a single insemination before transfer was adequate when fresh semen from fertile stallions was used. Absence of a preovulatory follicle did not appear to affect pregnancy rates in the present experiments. The transfer of sperm and oocytes (GIFT) into the oviduct was successful and repeatable as an assisted reproductive technique in the equine.

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 recipient's 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.

Use of oocyte transfer in a commercial breeding program for mares with reproductive abnormalities.

In some mares with lesions of the reproductive tract, embryo collection and survival rates are low, or collection of embryos is not feasible. For these mares, oocyte transfer has been proposed as a method to induce pregnancies. In this report, a method for oocyte transfer in mares and results of oocyte transfer performed over 2 breeding seasons, using mares with long histories of subfertility and various reproductive lesions, are described. Human chorionic gonadotropin or an implant containing a gonadotropin-releasing hormone analog was used to initiate follicular and oocyte maturation. Oocytes were collected by means of transvaginal ultrasound-guided follicular aspiration. Following follicular aspiration, cumulus oocyte complexes were evaluated for cumulus expansion and signs of atresia; immature oocytes were cultured in vitro to allow maturation. The recipient's ovary and uterine tube (oviduct) were exposed through a flank laparotomy with the horse standing, and the oocyte was slowly deposited within the oviduct. Oocyte transfer was attempted in 38 mares between 9 and 30 years old during 2 successive breeding seasons. All mares had a history of reproductive failure while in breeding and embryo transfer programs. Twenty pregnancies were induced. Fourteen of the pregnant mares delivered live foals. Results suggest that oocyte transfer can be a successful method for inducing pregnancy in subfertile mares in a commercial setting.

Comparison of culture and insemination techniques for equine oocyte transfer.

This study was designed to test 3 approaches for insemination and transfer of oocytes to recipient mares. Oocytes were recovered transvaginally from naturally cycling donor mares 24 to 26 h after an intravenous injection of 2500 IU of hCG when follicles reached 35 mm in diameter. Multiple oocytes (1 to 4) were transferred surgically into the oviducts of 4 or 5 recipient mares per group. Three groups of transfers were compared: 1) transfer of oocytes cultured in vitro for 12 to 14 h postcollection with insemination of the recipient 2 h postsurgery; 2) transfer of oocytes into the oviduct within 1 h of collection, with completion of oocyte maturation occurring within the oviduct, and insemination of the recipient 14 to 16 h postsurgery; and 3) transfer of spermatozoa and oocytes (cultured 12 to 14 h in vitro) into the oviduct. Numbers of embryos detected by Day 16 of gestation were not different (P>0. 1) for groups 1, 2, and 3 (57%, 43% and 27%). Therefore, equine oocytes successfully completed the final stages of maturation within the oviduct, and sperm deposited within the oviduct were capable of fertilizing oocytes.

Treatment with progesterone and 17 beta-oestradiol to induce emergence of a newly-recruited dominant ovulatory follicle during oestrus synchronisation with long-term use of norgestomet in Brahman heifers.

The aim of this study was to determine the effect on ovarian follicular growth and atresia, of acute treatment with either 100 mg of progesterone (n = 10), 200 mg of progesterone (n = 10), 10 mg of oestradiol + 100 mg of progesterone (n = 10), 10 mg of oestradiol (n = 10) or no treatment (n = 10), given on Day 10 of a 17-day treatment with a norgestomet implant in randomly cycling Bos indicus heifers. The fate of the dominant follicle on Day 10, emergence of the new cohort of follicles and the intervals from implant removal to ovulation were recorded by ultrasonography. Plasma concentrations of Luteinizing hormone (LH), progesterone and oestradiol were determined during the time when the norgestomet implant was in place. All treatments resulted in the emergence of a new cohort of follicles within 5 days of administration. The day of emergence of the ovulatory follicle tended to be delayed after treatment with 100 mg of progesterone (2.7 +/- 0.3 days after treatment), 200 mg of progesterone (3.7 +/- 0.5 days after treatment), 10 mg of oestradiol + 100 mg of progesterone (4.4 +/- 0.2 days after treatment) and 10 mg of oestradiol (4.6 +/- 0.4 days after treatment) compared to control heifers (1.4 +/- 1.4 days after time of treatment). The mean interval from implant removal to onset of oestrus was significantly shorter after treatment with 100 mg of progesterone (38.4 +/- 2.6 h) than after treatment with 200 mg of progesterone (61.5 +/- 3.9 h) but otherwise, the mean interval from implant removal to onset of oestrus did not differ. Oestrus synchrony, measured by the sample standard deviation of oestrus onset, was tighter in all treatment groups compared to untreated control heifers. The mean interval from implant removal to ovulation did not differ significantly between groups. The synchrony of ovulation, measured by the sample standard deviation of the interval from implant removal to ovulation, was significantly tighter after treatment with 100 mg of progesterone, 200 mg of progesterone and 10 mg of oestradiol compared to control heifers. Treatment with 10 mg of oestradiol resulted in ovulation in seven of 10 heifers before implant removal, three of which failed to ovulate after implant removal. Progesterone administered on Day 10 lowered plasma LH concentrations (P < 0.05), whereas treatment with oestradiol caused a surge of LH and ovulation. Progesterone administered with oestradiol prevented the LH surge. A combination treatment of oestradiol and progesterone given on Day 10 of a 17-day norgestomet treatment in a range of follicular states resulted in the consistent emergence of a new cohort of follicles which included the eventual ovulatory follicle.

Close synchrony of ovulation in superstimulated heifers that have a downregulated anterior pituitary gland and are induced to ovulate with exogenous LH.

The synchrony of ovulation was examined in superstimulated heifers that had a downregulated pituitary gland and which were induced to ovulate by injection of exogenous LH. The pituitary was downregulated and desensitized to GnRH by treatment with the GnRH agonist deslorelin. Nulliparous heifers (3.5 yr old) at random stages of the estrous cycle were assigned to 1 of 3 groups, and on Day -7 received the following treatments: Group 1 (control, n = 8), 1 norgestomet ear implant; Group 2 (GnRH agonist, n = 8); Group 3 (GnRH agonist-LH protocol, n = 8), 2 deslorelin ear implants. Ovarian follicle growth in all heifers was superstimulated with twice-daily intramuscular injections of FSH (Folltropin-V): Day O, 40 mg (80 mg total dose); Day 1, 30 mg; Day 2; 20 mg; Day 3, 10 mg. On Day 2, all heifers were given a luteolytic dose of PGF (7 A.M.), Norgestomet implants were removed from heifers in Group 1 (6 P.M.). Heifers in Group 3 were given an injection of 25 mg, i.m. porcine LH (Lutropin) on Day 4 (4 P.M.). Ovarian follicle status was monitored at 8-h intervals from Day 3 (8 A.M.) to Day 6 (4 P.M.) using an Aloka Echo Camera and 7.5 MHz transducer. Heifers in Groups 2 and 3 exhibited estrus earlier (P < 0.05) than heifers in Group 1. Heifers in Group 2 did not have a preovulatory LH surge and they did not ovulate. Individual control heifers in Group 1 ovulated between 12 A.M. on Day 5 and 8 A.M. on Day 6. Heifers with deslorelin implants and injected with LH in Group 3 ovulated between 4 P.M. on Day 5 and 8 A.M. on Day 6. It was confirmed that superstimulated heifers with GnRH agonist implants can be induced to ovulate with LH. It was also demonstrated that ovulation is closely synchronized after injection of LH. Thus, a single, fixed-time insemination schedule could be used in a GnRH agonist-LH superovulation protocol, with significant practical and economic advantages for superovulation and embryo transfer programs.

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist.

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.

Influence of the luteinizing hormone-releasing hormone agonist, deslorelin, on patterns of estradiol-17 beta and luteinizing hormone secretion, ovarian follicular responses to superstimulation with follicle-stimulating hormone, and recovery and in vitro development of oocytes in heifer calves.

The objective of this study was to determine the effects of inducing pituitary desensitization by treatment with an LHRH agonist (deslorelin) on reproductive hormone secretion and ovarian follicular status in heifer calves, before and during stimulation with FSH. The recovery and in vitro development of oocytes was also investigated. Brahman (Bos indicus) calves, 6 mo old, received either no treatment from Day 0 to Day 8 and injections of FSH on Days 9, 10, and 11 (controls, n = 10), or bioimplants of deslorelin on Day 0 and injections of FSH on Days 9, 10, and 11 (deslorelin calves, n = 10). Ovarian follicular characteristics were determined on Days -2, 0, and 8 by ultrasonography; follicle sizes (2-4 mm, 5-7 mm, 8-9 mm, or > or = 10 mm) were recorded. Ovaries were removed surgically on Day 12, surface follicle numbers and sizes were recorded, and oocytes were aspirated, graded (A-grade, B-grade, denuded, atretic), and prepared for in vitro fertilization and culture. Blood samples were taken throughout the experiment to monitor plasma concentrations of LH, estradiol-17 beta (estradiol) and progesterone. Treatment with deslorelin desensitized the pituitary in heifer calves and altered patterns of LH and estradiol secretion. There were no apparent consistent effects of deslorelin treatment on follicle numbers and growth. A higher number of combined A-grade and B-grade oocytes were obtained from heifers treated with deslorelin, which, in turn, resulted in twice the number of blastocysts. Treatment with an LHRH agonist provides a model for studying the hormonal requirements for follicle growth and in vivo oocyte maturation in heifer calves.