The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 μg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 μg/mL BSP1 (35.6 ± 2.5%) and 40 μg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma.

ABSTRACT. Embryo sexing is a powerful tool for livestock producers because it allows them to manage their breeding stocks more effectively. However, the cost of supplies and reagents, and the need for trained professionals to biopsy embryos by micromanipulation restrict the worldwide use of the technology to a limited number of specialized groups. The aim of this study was to couple a fast and inexpensive DNA extraction protocol with a practical biopsy approach to create a simple, quick, effective, and dependable embryo sexing procedure. From a total of 1847 sheep and cattle whole embryos or embryo biopsies, the sexing efficiency was 100% for embryo biopsies, 98% for sheep embryos, and 90.2% for cattle embryos. We used a primer pair that was common to both species and only 10% of the total extracted DNA. The whole protocol takes only 2 h to perform, which suggests that the proposed procedure can be readily applied to field conditions. Moreover, in addition to embryo sexing, the procedure can be used for further analyses, such as genotyping and molecular diagnosis in preimplantation embryos.

Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin-endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptusderived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR-Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2-derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally.Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2-derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep. K E Y W O R D Sconceptus, genome editing, implantation, prostaglandins, sheep CONFLICT OF INTERESTSThe authors declare that there are no conflict of interests. ORCID Eleanore V. O'Neil

Cloning by somatic cell nuclear transfer (SCNT) is characterized by low efficiency and the occurrence of developmental abnormalities, which are rather poorly studied phenomena in goats. This study aimed at comparing overall SCNT efficiency in goats by using in vitro-matured (IVM) or in vivo-matured oocytes and fibroblast donor cells (mock transfected, transgenic, or wild type), also characterizing symptoms of the Abnormal Offspring Syndrome (AOS) in development, comparing results with pregnancies produced by artificial insemination (AI) and in vivo-derived (IVD) embryos. The SCNT group had lower pregnancy rate (18.3%, 11/60), total number of concepti (20.0%, 12/60), term births (3.3%, 2/60), and live births (1.7%, 1/60) than both the IVD (77.8%, 7/9; 155.5%, 14/9; 122.2%, 11/9; 88.8%, 8/9) and the AI (71.4%, 10/14; 121.4%, 17/14; 100%, 14/14; 78.5%, 11/14) groups, respectively (p < 0.05). No SCNT pregnancies reached term using IVM oocytes, but in vivo-matured oocytes resulted in two term transgenic cloned kids. The proportion fetal membrane (FM) weight/birth weight reflected an increase in FM size and cotyledonary enlargement in clones, for disproportionally bigger newborns in relation to cotyledonary numbers. Overall, goat cloning showed losses and abnormality patterns similar to the AOS in cloned cattle and sheep, which have not been previously well recognized in goats.

Summary The present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen–thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

No abstract

No abstract

Although chronic renal disease is uncommon in horses, guidelines for management of the broodmare with kidney disease are needed to ensure successful foaling. Herein, we present a case report of a broodmare with chronic renal disease that produced a live foal, and parturition was predicted by monitoring pH and electrolytes in the milk. A 15year-old pregnant mare presented with a history of poor body condition and weight loss despite an excellent appetite. At presentation, the mare was bright, alert and responsive, with severe pitting ventral oedema and vital parameters within normal limits. Plasma biochemistry revealed azotaemia, hypercalcaemia and hypophosphataemia. Based on clinical ultrasonographic and biochemical parameters, the mare was diagnosed with renal failure. Pregnancy was assessed by transabdominal ultrasound, and the fetal parameters were within normal limits; however, an increased combined thickness of the uterus and placenta and oedematous fetal membranes were found. Monitoring of mammary secretions accurately predicted the onset of foaling at 326 days of gestation. A healthy filly was delivered, and the fetal membranes were passed in a timely manner without complications. Physical examination and haematological parameters were within the normal limits for a neonate foal, except for a mild elevation in blood urea nitrogen. Two weeks later, the mare was subjected to euthanasia due to worsening of her condition. At necropsy and on histopathological examination, the findings were consistent with chronic renal disease. This case demonstrates that a pregnant horse with chronic renal disease can be managed with supportive care and produce a viable foal. Fetal well-being and mare's prefoaling milk electrolytes were similar to parameters measured in healthy pregnant mares, suggesting that the feto-placental unit may be spared of the dam's systemic disease. Prepartum physiological changes in the milk may be unaltered with concurrent maternal renal disease and still be useful for foaling prediction.