In vitro performance and in vivo fertility of antibiotic-free preserved boar semen stored at 5℃

Background: Hypothermic preservation of boar semen is considered a potential method for omitting antibiotics from insemination doses, thereby contributing to the global antibiotic resistance defence strategy. The main challenges are chilling injury to spermatozoa and bacterial growth during semen storage leading to reduced fertility.Objectives: To examine chilling injury and the number and type of bacteria in boar semen stored at 5 ℃ in the absence of antibiotics, and to assess the applicability of hypothermic semen storage under field conditions.Material and methods: Boar ejaculates were extended with AndroStar~? Premium, stored at 17 ℃ with and at 5 ℃ without antibiotics and tested for functional sperm parameters by flow cytometry. Raw semen and extended samples were investigated bacteriologically. Fertility was evaluated after once-daily inseminations of 194 sows in a field study.Results: Lethal sperm damage assessed by motility and membrane integrity was low throughout storage in both experimental groups. Sublethal chilling effects based on the decrease of viable spermatozoa with low membrane fluidity were higher(P < 0.05) up until 72 h in sperm stored at 5 ℃ compared to 17 ℃ but did not differ after 144 h.After 72 h, incubation in capacitating medium for 60 min induced a similar decrease in viable sperm with high mitochondria membrane potential and low cytosolic calcium in both groups. In semen stored at 5 ℃, bacteria counts were below 103 CFU/mL and the bacteria spectrum was similar to that of raw semen. In 88% of 34 boars,cooled semen fulfilled the requirements for insemination. Fertility was high and did not differ(P > 0.05) between sow groups inseminated with semen stored antibiotic-free at 5 ℃ and semen stored at 17 ℃ with antibiotics.Conclusion: Despite subtle chilling effects and low bacterial numbers, antibiotic-free hypothermic storage of boar semen offers the possibility to reduce the use of antibiotics in pig insemination. However, strict sanitary guidelines must be maintained and further evidence of efficiency under field conditions is considered desirable.

Beneficial Effect of an Oral Antioxidant Supplementation (Fertimax2) on IVF-ICSI Outcomes: A Preliminary Clinical Study

Background: Considerable evidence points towards a significant role of oxidative stress (OS) in the pathogenesis of sperm dysfunction. OS as a result of an inappropriate balance between oxidants and antioxidants in the semen can cause DNA damage and lipid peroxidation leading to failure of conception, miscarriage or potentially even childhood cancer. The objective of this study was to investigate whether a male antioxidant therapy can improve semen parameters and the results of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Methods: A total of forty eight infertile couples were considered. Male participants were administrated Fertimax2 antioxidant treatment for at least two months prior to their partner’s IVF-ICSI cycle. Sperm parameters (volume, concentration, progressive motility) and the IVF-ICSI outcomes were compared before and after the antioxidant treatment. The primary outcome measures were oocyte fertilization, cleavage and good embryo quality rates;the secondary outcomes were biochemical pregnancies, clinical pregnancies and implantation rates. Results: The principal finding that emerged from this study was that antioxidant therapy resulted in significant improvements in fertilization (p = 00.2), cleavage (p = 0.004) and good-embryo quality (p = 0.002) rates accompanied by a marked increase in clinical pregnancy (18.7% versus 2.5%) and implantation (11.8% versus 1.02%) rates. No significant changes in routine sperm parameters were observed. Conclusion: The Fertimax2 antioxidant therapy appears to influence favorably chances of conception in couples undergoing assisted reproduction treatment (ART).

Transplanting embryonic stem cells onto damaged human corneal endothelium

AIM To investigate whether human embryonic stem cells(hESCs) could be made to attach, grow and differentiate on a human Descemet's membrane(DM).METHODS Spontaneously differentiated hESCs were transferred onto a human corneal button with the endothelial layer removed using ocular sticks. The cells were cultured on a DM for up to 15 d. The genetically engineered hESC line expressed green fluorescent protein, which facilitated identification during the culture experiments, tissue preparation, and analysis. To detect any differentiation into human corneal endothelial-like cells, we analysed the transplanted cells by immunohistochemistry using specific antibodies.RESULTS We found transplanted cells form a single layer of cells with a hexagonal shape in the periphery of the DM. The majority of the cells were negative for octamer-binding transcription factor 4 but positive for paired box 6 protein, sodium potassium adenosine triphosphatase(NaKATPase), and Zona Occludens protein 1. In four of the 18 trials, the transplanted cells were found to express CK3, which indicates that the stem cells differentiated into corneal epithelial cells in these cases. CONCLUSION It is possible to get cells originating from hESCs to become established on a human DM, where they grow and differentiate into corneal endothelial-like cells in vitro.

Two Spontaneous Pregnancies after Treatment with Ovocyplus^TMamong an Infertile Patient with Two Failed IVF: A Case Report

The role of oxidative stress in female reproduction is becoming increasingly important, as recent evidence suggests that it has been implicated in the pathology of infertility of both known and idiopathic origin. Although its efficacy has yet to be well established, supplementation with antioxidants is a new tool being developed in the therapeutic armamentarium for female-factor infertility. We present a case of a spontaneous viable pregnancy in a 37-year-old patient with a history of two consequent In Vitro Fertilization (IVF) failures, occurring after a 5-month antioxidant treatment. This report suggests that oral antioxidants supplementation in female patients may provide an alternative or adjunct to conventional fertility therapies and improve their chances of becoming pregnant.

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling （18% w/v raffinose, RS-C） were compared with spermatozoa vitrified using 0.25 M sucrose （SV） or 18% w/v raffinose （RV）. The motility, vitality, and DNA damage （TUNEL assay） of fresh control （FC） spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes （n = 267） were randomly assigned into three groups for insemination： RV （n = 102）, RS-C （n = 86）, and FC （n = 79）. The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility （P 〈 0.01） and vitality （P 〈 0.05） were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose （15 - 1.8% [SV] vs 26 - 2.8% [RV] and 27 - 1.2% [RS-C]; P 〈 0.01）. Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa （P = 0.0053）. This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.