The clinical use of intracytoplasmic sperm injection (ICSI) in horses usually involves the transfer of embryos into recipient mares, resulting in substantial cost increases. This is essential when subfertile mares are oocyte donors; but some donors are fertile, with ICSI compensating for limited or poor-quality spermatozoa. Fertile oocyte donors could carry pregnancies, eliminating the need for a recipient. We assessed the potential of using oocyte donors as recipients for their own ICSI-produced embryos during the same cycle. Donors in oestrus and with large dominant follicles were administered ovulation-inducing compounds to cause follicle and oocyte maturation. Maturing oocytes were collected, cultured and fertilised using ICSI. At 6 or 7 days after ICSI, developing blastocysts were transferred into respective donors’ uteri, and pregnancy rates were determined. Twenty follicles were aspirated from nine mares and 12 oocytes were collected. After ICSI, 10 of the 12 oocytes (83%) cleaved, and eight (67% of injected oocytes) developed into blastocysts for transfer. Five pregnancies resulted from the eight transferred embryos (pregnancy rate 62% per embryo and 42% per sperm-injected oocyte). Following this synchronisation regime, ICSI-produced embryos can be transferred into oocyte donors’ uteri during the same cycle, allowing donors to carry pregnancies after assisted fertilisation.

Prostaglandins have important physiological roles in marine invertebrates, including larval development and reproduction. The prostaglandin E concentration fluctuates during the ovarian development of crustaceans. The biosynthetic pathway of prostaglandin, however, has not been well studied in portunid crabs, including in the mud crab, Scylla olivacea. In this study, the aim was to investigate the presence of prostaglandin E synthase (PGES), enzyme that catalyzes the terminal conversion in the prostaglandin E2 (PGE2) biosynthesis, and its gene expression in the central nervous system (CNS) and ovary during ovarian maturation of S. olivacea. cDNA sequence encoding PGES was cloned from the S. olivacea ovary. The PGES transcript of S. olivacea (Scyol-PGES) consists of 1258 nucleotides, which encodes for 420 amino acid PGES protein precursor. Investigation of gene expression by RT-PCR indicated that Scyol-PGES was detected in all organs studied. Based on in situ hybridization, Scyol-PGES was detected in the I to III stages for oocyte development of Stage 3 of ovarian development, and in the CNS, including the various neuronal clusters of the brain. In the ventral nerve cord, the Scyol-PGES gene was expressed in the neurons within the subesophageal, thoracic and abdominal ganglia. The Scyol-PGES gene expression as indicated by relative abundance of mRNA in the Stage 4 of ovarian development was greater than that at Stages 1 to 3 of ovarian development. This is the first report on PGES in the mud crab, S. olivacea, and its gene expression suggested the involvement of PGES in the ovarian development of this species.

The purpose of this study was to evaluate the contribution of the total content of glycosaminoglycans (GAGs), cortisol, iron, and hemoglobin (Hb) in term and nursing mares. Twenty-four healthy mares, aged 7 ± 1.5 years, were examined. Eighteen pregnant mares were considered the observational group, and six nonpregnant mares were considered the control group, by random selection. Blood samples were taken at the 10 and 11 months of gestation and at 15 and 30 postpartum days for the observational group and in baseline condition for the control group. Two-way analysis of variance showed a significant interaction between peripartum, postpartum, and time points for GAGs (F = 34.75; P < .0001) and Hb (F = 8.77; P = .007) changes. A significant effect of peripartum (F = 64.96; P < .0001) on the Hb changes was observed, with higher concentrations at 15 days of postpartum (P < .01) than 11 months of pregnancy. Compared with control group, observational group showed lower Hb values along peripartum (P < .01) and at 30 days of postpartum (P < .01). A significant and positive correlation between GAGs and cortisol concentrations (r = 0.99; P = .008) was also shown. The present study contributes to a deeper knowledge on the dynamic endocrine–metabolic profile of mare throughout the transition from one to another physiological stage.

Cryopreservation impairs oocyte quality, which may be associated with abnormal gene expression. Currently, alteration of mRNA levels in vitrified porcine oocytes has not been well characterized. The aim of this study was to analyze transcriptome profiles with RNA sequencing (RNA-seq) in porcine immature oocytes and their surrounding cumulus cells (CCs) after vitrification and in vitro maturation (IVM). There were 19 upregulated and 18 downregulated genes differentially expressed in vitrified oocytes, with no significant GO enrichment or KEGG pathway identified for these genes. In addition, CCs derived from vitrified oocytes had 40 significantly upregulated and 100 significantly downregulated genes. In total, 7 GO terms were significantly enriched in molecular function and biological process, and only MAPK signaling pathway reached significant enrichment based on KEGG analysis. Moreover, selected differentially expressed genes had similar expression patterns through comparison between results from qRT-PCR and RNA-Seq. In conclusion, our data provided detailed information on mRNA transcriptomes in porcine immature oocytes and CCs after vitrification and IVM, which offered now insights regarding reduced developmental potential of the vitrified oocytes.

In all eutherian mammals, there are two modes for supplying nutrients to the conceptus, histotrophic and hemotrophic nutrition. In general, these modes are sequential; although in some species, the two operate in parallel throughout the entire pregnancy. Although hemotrophic nutrition is the most important for proper growth of the fetus as assessed by birth weight, there is growing evidence that the phase of histotrophic nutrition is critical for initial development of the placenta. Evidence from animal species has revealed the existence of a signaling dialog between the trophoblast of the placenta and the endometrial glands, whereby the secretion of nutrients, collectively known as uterine milk proteins, and growth factors is upregulated during early pregnancy. In this way, the placenta is able to stimulate its own development. Circumstantial evidence suggests that an equivalent dialog occurs in the human and that deficiencies in endometrial function in early pregnancy may underpin complications of later pregnancy. The conservation of the trophoblast-endometrial dialog across species, with such differences in placental types as the horse and the human, suggests that it is of fundamental importance in placentation. The implication is that attempts should be made to ensure the endometrium is in optimal condition before conception.

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll. Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte

In horses, determination of certain genetic traits/alleles (e.g. HYPP, SCID, sex, or color) before transfer of embryo often would be advantageous due to the costs of these pregnancies. An attractive option is pre-implantation genetic diagnosis, but to date, few biopsied equine embryos have resulted in pregnancies. In the current experiment, 10 embryos ranging in diameter from 160-575 μm were biopsied. To obtain embryos, donor mares were monitored using transrectal ultrasonography. When a follicle >35 mm in diameter was observed, 2500 IU of hCG or 1.5 mg of deslorelin acetate was administered, and mares were inseminated daily until ovulation was detected. Embryos were recovered nonsurgically on Days 6.5-7 (Day 0 = ovulation). Trophoblast biopsies were collected in a 30-μL droplet of Syngro Holding Media (Bioniche, Belleville, Ontario, Canada) using a Piezo drill and beveled injection pipette (7-10 μm outer diameter). Biopsy samples ranged from 4-10 cells by visual assessment. After removal of the embryo, the droplet containing the biopsied cells was moved into an Eppendorf tube and centrifuged at 11 000g for 10 min. Supernatant was removed leaving 5 μL of sample, which was snap frozen for later genetic testing. Embryos were immediately transferred nonsurgically into the uteri of recipient mares synchronized to ovulate 0 to 2 days after the donor. Pregnancy examinations were performed via ultrasonography on Days 11, 12, 14, 16, 20, and 25. Pregnancies were confirmed on Day 16 for 6 of the 10 transfers. Five embryos (175-240 μm) had confirmed heartbeats on Day 25; one recipient mare lost her pregnancy (160 μm) between Days 16 and 20. The two largest embryos, 390 and 575 μm, did not result in pregnancies. As a preliminary experiment, 7 embryos (150-300 μm) were vitrified according to Eldridge-Panuska et al. (2005) after biopsying and later warmed and transferred directly. Three (all 200 μm) Day 16 pregnancies resulted from the transfer of vitrified, biopsied embryos. Two pregnancies were maintained to a heartbeat; one pregnancy resulted in the formation of an empty trophoblastic vesicle. All pregnancies were terminated on or after Day 25 to collect embryos for further genetic testing. For pre-implantation genetic testing, a duplex-nested PCR was developed for amplification of the DNA from the biopsied cells using primers for sex chromosome-linked zinc finger protein genes (ZFX/ZFY; 445 bp), and 2 pairs of primers for equine-specific sex-determining region on the Y-chromosome (SRY; 217 bp, 121 bp). Experiments on XX and XY genomic DNA from white blood cells revealed accurate genetic testing on as little as 9 pg of DNA, which equals 1 cell. Preliminary results revealed successful sex genotyping on biopsied material, and current experiments are underway to confirm and expand on these results.

Young (4 to 9 yr) and old (>or=20 yr) mares were treated with equine follicle-stimulating hormone (eFSH), and oocytes were collected for intracytoplasmic sperm injections (ICSI). Objectives were to compare: (1) number, morphology and developmental potential of oocytes collected from young v. old mares from cycles with or without exogenous eFSH and (2) oocyte morphology parameters with developmental competence. Oocytes were collected from preovulatory follicles 20 to 24 h after administration of recombinant equine LH and imaged before ICSI for morphological measurements. After ICSI, embryo development was assessed, and late morulae or blastocysts were transferred into recipients' uteri. Cycles with eFSH treatment resulted in more follicles (1.8 v. 1.2) and more recovered oocytes (1.1 v. 0.8) than those without eFSH. Age and eFSH treatment did not effect cleavage, blastocyst and pregnancy rates. Treatment with eFSH had no effect on oocyte morphology, but age-associated changes were observed. In old mares, zona pellucidae (ZP) were thinner than in young mares, and perivitelline space and inner ZP volume (central cavity within the ZP) were larger and associated with oocytes that failed to develop. These results suggest that administration of eFSH can increase the number of oocytes collected per cycle. Oocyte morphology differed with age and was associated with developmental competence.

Intracytoplasmic sperm injection (ICSI) can be used to produce offspring from mares or stallions with fertility problems. Early embryos can be transferred into recipients’ oviducts or embryos can be cultured for nonsurgical transfer into recipients’ uteri. The aim of this research was to evaluate the optimal time to transfer ICSI-produced embryos into recipients’ uteri. The objective was to compare pregnancy rates after the nonsurgical transfer of early morulae, compact morulae, and blastocysts. Oocytes were collected by transvaginal, ultrasound-guided follicular aspirations between 20 and 24 h after administration of deslorelin (1.5 mg, i.v., Francks Pharmacy, Ocala, FL, USA) to donors. Oocytes were cultured for 16 to 18 h in M199 (Invitrogen, San Jose, CA, USA) with 10% FCS (HyClone, Logan, UT, USA), 0.2 mm pyruvate (Sigma, St. Louis, MO, USA) and 25 μg mL–1 gentamycin (Sigma, St. Louis, MO, USA) at 38.5°C and 6% CO2. Cumulus cells were denuded by gentle pipetting, after oocytes were placed into hyaluronidase (500 U mg–1, Sigma, St. Louis, MO, USA). Oocytes were injected with a single sperm from one of two stallions, with sperm being frozen or sex-sorted and refrozen (Squires EL et al. 2008). Forty-six of 62 (74%) injected oocytes cleaved. The presumptive zygotes were cultured in DMEM/F12 (Sigma, St. Louis, MO, USA) with 10% FCS at 38.5°C and an atmosphere of 5% CO2, 5% O2 and 90% N2. Embryos were placed in fresh medium every 3 days. Injected oocytes were observed for cleavage at 2 days, and embryos were assigned to a transfer group. Embryos were transferred as early morulae (EM, 8-cell to precompaction stage, n = 14), compact morulae (CM, postcompaction, n = 10) or blastocysts (B, observed blastocoele, n = 9) into recipients at 3 to 5 days (EM), 3 to 6 days (CM) or 5 to 6 days (B) after the recipient’s ovulation or follicle aspiration. Pregnancy scans were performed on Day 12, 14, and 16 after ICSI, and pregnant recipients were examined until 30 days to detect the embryo proper and heartbeat. Number of embryonic vesicles detected per transferred embryo was determined by Fisher’s Exact Test. Pregnancy rates differed (P = 0.0017) among groups (EM, 1/14, 7%; CM, 4/10, 40%; B, 7/9, 78%), with fewer (P = 0.001) EM than B resulting in embryonic vesicles; however, pregnancy rates were not significantly different between CM and other embryo stages. An embryo proper with heartbeat was observed for all pregnancies, with the exception of one pregnancy resulting from the transfer of a blastocyst. In this study, all blastocysts were transferred prior to the embryo attempting to hatch from the zona pellucida, but after the appearance of a distinct blastoceole. In our study, pregnancy rates were higher after the transfer of later v. earlier stages of embryo development.

Obtaining adequate numbers of sex-selected sperm for uterine inseminations can be difficult, especially if fresh semen is not available or sperm numbers are limited. Intracytoplasmic sperm injection (ICSI) requires minimal sperm numbers and can be used for the assisted fertilization of oocytes. The objectives of this study were to: (1) thaw, sex-sort, and refreeze semen, and (2) determine if cleavage rates of oocytes were similar after ICSI using frozen v. frozen, thawed, sex-sorted, and refrozen sperm. The final aim was to produce foals by ICSI using sex-sorted sperm. Light-horse mares between 3 and 15 years were used as oocyte donors. When a follicle 30 to 35 mm and endometrial edema was imaged using ultrasound, deslorelin (1.5 mg, i.m., Franck’s Pharmacy, Ocala, FL, USA) was administered to induce follicular maturation. Between 20 and 24 h after deslorelin, oocytes from preovulatory follicles were collected by transvaginal, ultrasound-guided follicular aspirations. Oocytes were cultured for 16 to 18 h in TCM 199 with 10% FCS, 0.2 mm pyruvate at 38.5°C and in an atmosphere of 6% CO2. Semen from a single ejaculate of two stallions was frozen for the experiment. Some of the frozen semen (control) was thawed and used for ICSI. Sperm from other straws were thawed, sex-sorted by flow cytometry, treated with cholesterol-loaded cyclodextrin, and refrozen in a skim milk-egg yolk diluent containing 0.52 m dimethyl formamide before thawing for ICSI. Sperm were thawed by cutting a thin section of a straw under liquid nitrogen; the straw section was dropped directly into medium at 38.5°C. Sperm were incubated for 10 min, before 1 μL of supernatant was removed and placed into a 5 μL drop of medium with 5% polyvinylpyrrolidone. From the droplet, sperm with progressive motility and normal morphology by visual inspection were selected for ICSI. Injected oocytes were placed in DMEM/F12 with 10% FCS for 48 h (±2 h) before assessment of cleavage. Numbers of cleaved per injected oocytes were compared by Fisher’s Exact Test and were lower (P < 0.001) for sex-sorted, refrozen sperm than for frozen control sperm (6/20, 30% and 15/18, 83%, respectively). At the completion of the project, three additional oocytes were injected with X-bearing sperm. The injected oocytes cleaved and developed into embryos under culture conditions [DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) with 10% FCS at 38.5°C and an atmosphere of 5% CO2, 5% O2 and 90% N2]. The resulting morula, blastocyst, and expanded blastocyst were transferred nonsurgically into recipients’ uteri. Pregnancies were established in recipients receiving the morula and expanded blastocyst, and two fillies were born in July 2008. Both foals appeared normal at birth; however, one foal became septic and was euthanized before 2 weeks of age. In this study, frozen, thawed, sex-sorted, and refrozen sperm were successfully incorporated into an ICSI program to produce early-stage embryos and sex-selected foals.

Reduced fertility in aged mares is associated with delayed early embryo development and lower pregnancy rates, potentially related to oocyte developmental competence. Human oocyte morphology has been associated with developmental potential, although comparative evidence is lacking in the mare. Exogenous FSH may be beneficial in obtaining more oocytes; however, effects on oocyte morphology and competence are unknown. Objectives were to determine if zona pellucida thickness (ZPT), ooplasm volume (OV), and perivitelline space volume (PVSV) were related to mare age or FSH treatment and to cleavage, blastocyst, and pregnancy rates after intracytoplasmic sperm injection (ICSI). Cycles with and without eFSH treatment were alternated; eFSH treatments began in diestrus with a cohort of follicles ≥20 mm. Oocytes were collected by transvaginal aspiration from follicles >30 mm from young (4 to 9 years) and old (>20 years) mares at 20 to 24 h after administration of recombinant eLH. Oocytes were cultured for 18 h in TCM-199 at 38.5°C in 6% CO2 in air. Sperm were injected 40 ± 1 h after eLH, using frozen sperm from a single ejaculate. Presumptive zygotes were incubated in Dulbecco's modified Eagle's medium/F12 + 10% fetal calf serum at 38.5°C in 5% CO2, 5%O2, and 90% N2. Cleavage (≥2 cells) was recorded 48 h after ICSI. Blastocysts considered viable (formation before 9 d and good quality) were transferred nonsurgically into recipients 3 to 7 days after ovulation. Only pregnancies of fetuses with heart beats were included. Morphological parameters of oocytes (old, n = 40; young, n = 37) were obtained from photographic images taken at ICSI and analyzed by computer-assisted measurement using digital calipers (Spot Software, Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Zona pellucida thickness was averaged from 2 measurements 90° to 180° apart. Ooplasm volume was calculated (4/3πr3) from the average of 2 diameters of the ooplasm 90° apart; and PVSV was calculated as the difference of the vitelline membrane volume and that of the volume at the inner volume of the ZP calculated as an oblate spheroid (4/3πa2b) from the average of 2 diameters. Zona pellucida thickness, OV, and PVSV were analyzed using 2-way ANOVA for main effects of age and treatment and 3-way ANOVA by adding cleavage as a factor. Zona pellucida thickness was less (P = 0.007) for old compared with young (least squares mean SEM of 11.4 ± 0.2 and 12.3 ± 0.2 µm, respectively) with no effect on cleavage, blastocyst, or pregnancy rates. Ooplasm volume was not different (P = 0.14) between old and young (309 036 ± 5373 and 320 544 ± 5639 µm3, respectively) and did not affect cleavage, blastocyst, or pregnancy rates. The PVSV was greater (P = 0.001) in old compared with young (157 505 ± 10 853 and 102 161 ± 11 388 µm3, respectively) and may be related to the lower cleavage (P = 0.03), blastocyst (P = 0.02), and pregnancy (P = 0.05) rates. Treatment with FSH had no effect (P > 0.1) on morphology or embryo development. In this study, ZPT and PVSV differed with mare age and could be of predictive value for oocyte developmental competence.

Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2±0.4 yr of age (mean±S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0±0.1h after administration of hCG to estrous donors and cultured 16.4±0.2h prior to transfer into recipients’ oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P>0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors <20 than ≥20 yr (mean ages±S.E.M. of 15.5±0.4 and 23.0±0.3 yr, respectively) tended (P=0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer.