Inhibiting diacylglycerol acyltransferase-1 reduces lipid biosynthesis in bovine blastocysts produced in vitro.

Diacylglycerol acyltransferase-1 (DGAT1) is one of the DGAT enzymes that catalyzes the final step in the synthesis of triacylglycerol, which is a major component of the lipid droplets in embryos. Intracellular lipids accumulated in embryos produced in vitro have been associated with reduced cryotolerance and quality. The objective of the present study was to investigate the influence of DGAT1 inhibition on embryo development, quality, and post-vitrification survival, in addition to expression profiles of selected lipid metabolism-regulating and oxidative stress genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro and were cultured in synthetic oviduct fluid (SOF) supplemented with 5% fetal calf serum (FCS) alone (Control) or with 1, 5, 10 or 50 µM DGAT1 inhibitor (A922500®; D1, D5, D10, and D50, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for DGAT1 inhibitor dilution) from 54 h post-insemination until Day 8 post insemination. No differences were found in blastocyst yield on days 7 and 8 in Control, CDMSO, D10, and D50 groups. Embryos cultured with 10 or 50 µM DGAT1 inhibitor had greater mitochondrial activity (P < 0.01), and increased number of cells (P < 0.05), while the cytoplasmic lipid content was reduced (P < 0.01), the latter associated with altered expression profiles of selected genes regulating lipid metabolism or genes related with oxidative stress (transcript abundance increased for SLC2A1 and SLC2A5 and decreased for DGAT1 and GPX1). Importantly, the survival rate of blastocysts produced with 10 µM DGAT1 was higher than that of Control, CDMSO and D50 groups at 72 h after vitrification and warming (73.8 vs 57.1, 55.9 and 56.1%, respectively, P < 0.001). In conclusion, inhibition of DGAT1 synthesis in bovine embryos produced in vitro abrogates the negative effect of FCS by decreasing their lipid content, increasing mitochondria activity and improving embryo cryotolerance, as well as favoring the expression of lipid metabolism regulating and oxidative stress-related transcripts.

Fertilizing capacity of vitrified stallion sperm assessed utilizing heterologous IVF after different semen warming procedures.

The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 µm/s) and ALH (3.00 ± 0.2 µm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment 2, the fertilizing capacity of vitrified and frozen sperm was assessed utilizing heterologous IVF procedures, using cattle oocytes. Vitrification resulted in greater values (P < 0.05) than freezing for the number of bound sperm (1.36 ± 0.3 and 0.69 ± 0.2, respectively). There were no differences between frozen or vitrified sperm in pronuclear formation (26 hours post-insemination - hpi; 14.08 ± 4.2 % and 22.78 ± 4.8 %, respectively) or cleavage rate (32.77 ± 4.3 % and 39.66 ± 4.6 %, respectively). In conclusion, vitrified stallion sperm warmed in a water bath at 60 ºC had the capacity to penetrate cattle oocytes, leading to pronuclear formation and hybrid embryo cleavage after heterologous IVF.

Influence of sperm filtration and the addition of glycerol to UHT skimmed milk- and TEST-based extenders on the quality and fertilizing capacity of chilled ram sperm.

The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, µm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96 h of chilling were higher when the UHT extender (P < 0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96 h compared to all other treatments (P < 0.01). The best results of all were obtained with non-filtered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24 h (CS24) or chilled-for-48 h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3 ±â€¯3.2; 48.8 ±â€¯3.2; and 43.3 ±â€¯3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (P > 0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96 h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48 h.

Fertilizing capacity of vitrified epididymal sperm from Iberian ibex (Capra pyrenaica).

In this study, we successfully described for the first time a vitrification of epididymal Iberian ibex spermatozoa. Spermatozoa from epididymis were obtained from 15 Iberian ibex. The right epididymis' semen sample was vitrified and the left one was frozen. After thawing/warming, samples were selected by density gradient. Sperm characteristics from each treatment were evaluated. To test the spermatozoa fertilization ability, heterologous IVF was carried out using bovine oocytes. Despite of the observation of a decrease of about 40% for motility sperm between pre-freezing and post-thawing (75.0 ±â€¯5.2 and 45.0 ±â€¯6.0) and pre-vitrification and post-warming (78.2 ±â€¯5.2 and 33.9 ±â€¯6.2) (P < .05), after the washing, an improvement of sperm motility was found when using the vitrification treatment compared to frozen-thawed. Heterologous IVF showed that Iberian Ibex spermatozoa, either frozen-thawed or vitrified-warmed, were equally capable of penetrating ZP intact bovine oocytes, leading to pronuclear formation (%) and hybrid embryo cleavage (%), (31.3 ±â€¯27.2 and 45.1 ±â€¯24.4, respectively). As expected, in the homologous IVF group, higher percentages of penetration, pronuclei formation and cleavage were found compared to heterologous groups using Iberian ibex frozen and vitrified sperm (P < 0,5). The highest pronuclei formation was found after 20 h post insemination in both heterologous IVF groups (30.2 ±â€¯6.7 and 31.7 ±â€¯21.5 thawed and vitrified group). Consequently, the cleavage rate (48 h) followed the same results to homologous and thawed and vitrified groups (76.1 ±â€¯15.9; 31.3 ±â€¯27.2 and 45.1 ±â€¯24.4, respectively) (P < .05). In conclusion, Iberian ibex sperm vitrification is a promising and useful alternative to conventional methods resulting in good quality spermatozoa post-thaw, and an adequate in vitro fertilizing ability.

Exocannabinoids effect on in vitro bovine oocyte maturation via activation of AKT and ERK1/2.

Endocannabinoids are known to mediate practically all reproductive events in mammals; however, little is known about their role in oocyte maturation. Through RT-PCR and immunocytochemistry, this study confirms the presence of CB1 and CB2 cannabinoid receptors in bovine oocytes and shows how exposure to the exogenous cannabinoids HU-210 and THC during their in vitro maturation (IVM) activates the phosphorylation of AKT and ERK1/2 proteins associated with the resumption of meiosis. Although supplementation with HU-210 or THC during IVM did not increase blastocyst yields, the expression of interferon tau (IFNτ) and gap junction alpha-1 protein (GJA1) was enhanced at the blastocyst stage. Our data suggest that cannabinoid agonists may be useful IVM supplements as their presence during oocyte maturation upregulates the expression in blastocysts of key genes for embryo quality.

Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa.

Assisted reproductive technologies are of great importance for increasing the genetic diversity in captive animals. The use of bovine or murine oocytes in heterologous IVF provides advantages compared to homologous IVF in nondomestic animals, such as the accessibility to oocytes and the availability of well-developed in vitro maturation systems. The aim of this study was to determine the heterologous IVF parameters using cryopreserved dolphin spermatozoa and zona-intact bovine or murine oocytes and to examine the nuclear chromatin status of the dolphin spermatozoa. All the processes involved in the fertilization including embryo cleavage were observed by confocal microscopy and hybrid embryo formation was confirmed by polymerase chain reaction. Heterologous bovine IVF showed no polyspermy, lower percentages of pronuclear formation, and a lower cleavage rate compared to homologous IVF group (34.8% vs. 89.3%). Heterologous murine IVF showed a lower cleavage rate than homologous IVF (9.6% vs. 77.1%). With respect to dolphin sperm chromatin, it was more stable, i.e. more resistant to EDTA-SDS decondensation than the bovine sperm chromatin. This study revealed the stability of the dolphin sperm chromatin and the ability of the dolphin spermatozoa to penetrate zona-intact bovine and murine oocytes, leading to hybrid embryo formation.

The effect of bovine embryo culture without proteins supplements until day 4 on transcription level of hyaluronan synthases, receptors and mtDNA content.

The effect of bovine embryo culture on a flat surface, (without a surface-active compound) on the level of mRNA expression of hyaluronan (HA) synthases (Has1, Has2 and Has3), Ha receptors RHAMM and C44 receptors was evaluated by mitochondrial DNA concentration and in vitro development. Cultures were evaluated up to 96 h post-insemination (hpi) using SOFaa medium. Of the three Has isoforms, Has2 expression only increased in the bovine serum albumin (BSA)-only supplemented groups regardless of time of BSA addition. Expression of RHAMM receptors was highly dependent on the addition of HA, irrespective of the presence of BSA in the medium. In contrast, expression of the CD44 receptor gene was not affected by any treatment. The cleavage rates and number of embryos that developed to

Effects of hyaluronan, BSA, and serum on bovine embryo in vitro development, ultrastructure, and gene expression patterns.

Effects of hyaluronan (HA), BSA, and FCS on in vitro development, ultrastructure, and mRNA transcription of four developmentally important genes: apoptosis (Bax), oxidative stress (SOX), growth factor (IGF-II), and cell-to-cell adhesion (Ecad) were examined. Two biological origin HA, Hylartil and Hyonate and one produced by fermentation (f-HA) MAP-5 were tested. Embryos were cultured in SOF medium with 0.4% BSA or with 0.4% BSA and 10% FCS. HA was added 96 hr post insemination (pi) to half of the embryos from each culture group. Embryo development was not affected by either HA preparation, however, hatching rates were higher in Hyalartil and MAP-5 than in control and Hyonate (P < 0.05). There was no effect of HA on number of blastocysts developed in SOF + BSA. However, more blastocysts developed in SOF + BSA + f-HA than in SOF + BSA + FCS or with BSA + FCS + f-HA. HA added to SOF + BSA, increased level of expression of epidermal growth factor (EGF)-II and decreased the levels of expression of BAX, SOX, and Ecad (P < 0.05). Presence of FCS increased the levels of SOX and decreased the level of IGF-II (P < 0.05) and the addition of f-HA to SOF containing FCS showed no effect on the level of transcription of any analyzed genes. The fine structure of embryos cultured with f-HA irrespective of protein sources used was clearly improved. In summary, f-HA added 96 hr pi to SOF supplemented with BSA but not FCS improved development, molecular composition and fine structure of bovine embryos.