Evidence has accumulated in mammals suggesting a positive role for epidermal growth factor (EGF) as an inducer of oocyte maturation. The potential use of EGF as inducer of cytoplasmic and nuclear maturation was tested in women with > 10 oocytes retrieved in in-vitro fertilization (IVF), since we have previously observed that such oocytes are immature. Oocytes from 17 high responders were randomly allocated to one of the three treatment groups upon retrieval: control receiving no EGF (n = 93), 1.0 ng/ml EGF (n = 92) and 10.0 ng/ml EGF (n = 77) for 6 h before insemination. The rates of fertilization were respectively 54.6, 59.0, and 46.1%, suggesting that EGF is not effective at this maturational stage after this length of exposure. Embryo development was further analysed by the appearance of the embryos under the dissecting microscope and the number of blastomeres developed 48 h after insemination. No difference between groups was observed considering the number of blastomeres developed. However, embryos derived from oocytes treated with 10 ng/ml EGF displayed a worse appearance under the microscope. It is concluded that a 6 h incubation with EGF does not seem to affect cytoplasmic maturation in oocytes obtained after gonadotrophin treatment, as ascertained by the rate of fertilization following oocyte insemination.

The objective of this study was to investigate the immunocastration using against gonadotropin-releasing factor (GnRF) with Bopriva® (Zoetis) on the performance of production variables, meat quality, docility, and scrotal circumference in young rams, from Sheep production center “Tupac Amaru”, Junin region, Perú. A total of 60 animals were divided into four treatments: T1, T2, and T3 with 0.50 mL, 0.75 mL, and 1.0 mL of doses with Bopriva®, respectively, and T4 (control - 1.0 mL of placebo (distilled water)), with fifteen animals in each. The variables were measured every 15 days for three months (September to November 2018). Datasets were assessed by one-way ANOVA and subsequent posthoc Tukey´s test to detect significant differences among treatments. The results indicated differences found between treatments with the immunocastration compared with non-castrated animals for final weight, carcass weight and performance, scrotal circumference, and docility. Growth performance and meat quality characteristics were not adversely affected by immunocastration. Likewise, immunocastration with Bopriva proved to be effective to stop scrotal development and consequently reduces the sexual and aggressive behavior of young rams. The dose of 0.5 mL seems to be adequate due showed better performance for most variables compared to other treatments

Parthenogenetic embryos allow study of the roles of paternal and maternal genomes in early mammalian development. Nevertheless, pregnancies established with parthenotes arrest around 48 days. Genomic imprinting alterations, fewer cells, and apoptotic index are higher in parthenotes than in IVF embryos and are likely to contribute to the failure to reach full-term development. Neurotrophins are a family of anti-apoptotic cytokines that mediate survival, growth, and differentiation by binding to two types of cell surface receptors, tyrosine kinase (Trk) and the low affinity p75 neurotrophin receptor (p75). Trk and p75 receptors have been localized in early bovine in vitro-produced embryos. At present there are no available data on expression of Trk and numbers of cells in the inner cell mass (ICM) and the trophectoderm (TE) of parthenogenetic embryos. The aim of this study was to evaluate the quality of bovine parthenotes in terms of cell allocation and blastocyst development, and to analyze TrkA, TrkB, and TrKC expression in the ICM and TE. Starting from in vitro-matured slaughterhouse oocytes, embryos were produced by conventional IVF, while parthenotes resulted from ionomycin activation followed by 6-dimethylaminopurine. Zygotes were cultured in SOF + 6 gL– 1 BSA. In vitro development was assessed for IVF embryos on (and referred to on) Days 3, 6, 7, and 8 after fertilization, and 24 h before these time points for parthenotes. Data were analyzed by the GLM procedure of SAS SAS Institute, Inc., Cary, NC, USA). Parthenotes cleaved at rates similar to IVF embryos (80.8 � 3.9 v. 85.8 � 3.9, respectively), but percentages of 5–8 and 8–16 cell stages were lower in parthenotes (40.4 � 4.3 v. 67.9 � 4.3, P < 0.005, and 9.7 � 3.5 v. 25.3 � 3.5, P < 0.01, respectively). However, parthenogenetic blastocyst rates were higher than those in IVF embryos (Day 6: 33.6 � 2.6 v. 11.0 � 2.6, P < 0.005; Day 7: 49.2 � 4.1 v. 30.0 � 4.1, P < 0.02). Double staining showed fewer TE cells in parthenotes (78.7 � 8.5) than in IVF embryos (111.0 � 8.6, P < 0.02). This reduction accounted for a reduced number of total cells in parthenotes (105.3 � 9.9) v. controls (144.0 � 9.8, P < 0.01), while numbers of cells in the ICM were comparable (27.9 � 3.5 v. 31.1 � 3.5, in parthenotes and controls, respectively). As in the case of IVF embryos, immunocytochemical analysis showed positive staining for Trk receptors in parthenotes. Although parthenotes showed blastocyst development rates higher than in IVF embryos, the reduced amount of TE cells in parthenotes could negatively affect implantation. Interestingly, parthenotes do not contain abnormally reduced cell numbers in their ICM, and they express Trks. Therefore, specific stimulation of these receptors with appropriate cytokines could improve blastocyst development and embryonic stem cell derivation. This work was supported by the Spanish Ministry of Science and Education (AGL2005-04479). Dr. Muñoz was supported by FICYT.

Artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, is expressed in human and mice pre-implantation embryos and reproductive tract (Li et al. 2009 FEBS Lett. 583; Kawamura et al. 2012 PLoS One 7). In mice, artemin promotes in vitro embryo development and decreases apoptosis (Li et al. 2009 FEBS Lett. 583). The presence of artemin in cattle embryos and endometrium, however, is unknown. In this work we analysed artemin expression in bovine blastocysts and endometrium by immunohistochemistry and in uterine fluid (UF) by Western blot (WB). Briefly, Day-6 in vitro-produced (IVP) embryos (n = 50) were nonsurgically transferred to the uterus of heifers (n = 10, 50 IVP embryos per heifer) at nonconsecutive oestrus cycles. On Day 8, embryos and their corresponding diluted UF were flushed; blastocysts that developed entirely in vitro were also collected. In addition, endometrial samples were collected on Day 8 from slaughtered females that were embryo transferred (n = 6) and sham transferred (n = 6) on Day 5. Artemin localization was investigated in blastocysts and in endometrial samples, using immunohistochemical staining methods described elsewhere (Muñoz et al. 2012 J. Proteome Res. 11; Gómez et al. 2014 Reproduction pii: REP-14–0304). The signal-strength comparisons between uterus-exposed and IVP blastocysts were analysed using the software Confocal Uniovi Image-J. Quantification of WB protein bands was achieved by computer-assisted densitometry using Image-J software. Artemin was detected, with similar intensity, in the inner cell mass and trophectoderm from both uterus-developed and IVP blastocyst. All embryos analysed expressed artemin. The signal intensity and staining pattern observed did not differ between uterus-exposed and IVP blastocysts. In the endometrium, the most intense staining for artemin was localised to the apical sites in the luminal epithelium and in the glandular epithelium of superficial glands. There was also diffuse staining in the stroma and deep uterine glands. The uterine region and pregnant or cyclic status did not affect the artemin staining pattern. Artemin was detected by WB in all UF samples analysed (embryo transferred N = 10, sham transferred N = 10). However reliable quantitation of artemin by WB was unfeasible due to the broad dynamic range of artemin expression through samples. In conclusion, our results demonstrate the presence of artemin in bovine uterine endometrium and UF, and embryos during early development. As shown in mice, it is feasible that artemin might exert an autocrine/paracrine role during early embryo development in the cow. The study received grant support: Spanish Ministry of Science and Innovation (MINECO, project AGL2012–37772 and FEDER). M. M. was supported by grant MICINN-RYC08–03454. The authors are members of the COST Action FA1201 Epiconcept.

The production of sex-known offspring is a main objective in reproductive biotechnology. It has been reported that bovine ova developed in follicles with high concentrations of testosterone in vivo yielded significantly more male embryos in vitro (Grant V et al. 2008 Biol. Reprod. 78, 812–815). In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in fully in vitro conditions. Immature bovine cumulus–oocyte complexes (COCs; n = 750) from slaughterhouse ovaries were cultured in 199 HNaCO3 with polyvinyl alcohol (PVA) 0.1 mg mL–1 as a basic medium. Culture was made in two steps, a 24 h meiotic arrest (roscovitine 25 μm), and a subsequent in vitro maturation period with FSH-LH for 24 h. Testosterone (T-86500, Sigma-Aldrich, St. Louis, MO, USA) was added throughout the entire oocyte culture at 0, 30, 300, and 1500 nm. After in vitro fertilization (Day 0), zygotes were freed of cumulus cells by pipetting, and subsequently cultured in SOF + 6 g L–1 BSA up to Day 3. At this time, embryo development was recorded, and all embryos having 3 or more cells were treated with pronase to remove the zona pellucida. Zona-free embryos were washed in PBS containing PVA 0.1 mg mL–1 and individually frozen at –80°C until sex analysis by PCR (Bermejo-Alvarez P et al. 2008 Biol. Reprod. doi:10.1095/biolreprod.108.070169). A total of 252 embryos from 5 replicates were sexed. Data for development and sex-ratio are presented as % LSM ± SD. There were no interactions between testosterone treatment, embryonic sex, and embryonic stage analyzed. Testosterone did not affect development rates (P > 0.05) at any stage: cleavage (47.8 ± 6.8, 56.5 ± 6.8; 50.9 ± 6.8; 62.2 ± 6.8), 3 to 4 cells (40.6 ± 5.2, 45.8 ± 5.2; 37.8 ± 5.2; 47.7 ± 5.2) and >5 cells rates (24.5 ± 4; 27.3 ± 4; 21.3 ± 4; 25.3 ± 4) for 0, 30, 300, and 1500 nm testosterone, respectively. Cumulative percentages of male embryos were as follows: 53 ± 8 (n = 56), 42.6 ± 8 (n = 52), 53.6 ± 6 (n = 81) and 57.6 ± 8 (n = 63) for 0, 30, 300, and 1500 nm groups respectively (P > 0.05). These results show that the testosterone effects on oocyte ability to select Y-chromosome bearing spermatozoa are not reproducible in vitro under the present experimental conditions. Grant support: MEC, project AGL2008-01530; RTA2008-0082; M. Muoz is supported by FICYT.