Calcium Sensing Receptor (CaSR) is a G-protein coupled receptor which senses extracellular calcium and activates diverse intracellular pathways. The objective of our work was to demonstrate the presence of CaSR in bovine gametes and its possible role in fertilization and embryo development. The location of CaSR was demonstrated by immunofluorescence in bovine gametes; additionally bovine sperm were incubated with 5, 10 and 15 μM of the specific CaSR inhibitor NPS2143 in a Tyrode's Albumin Lactate Pyruvate medium (4 h). Sperm viability was maintained for all concentrations tested while total motility decreased significantly at 10 and 15 μM. Addition of 15 μM of NPS2143 during oocyte in vitro maturation did not alter the maturation rate. When NPS2143 (15 μM) was added to the fertilization medium during sperm-oocyte co-incubation the cleavage, morula and blastocyst rates remained unchanged. To confirm if 15 μM of NPS2143 exerted any effect on embryo development, NPS2143 was added to the embryo culture medium. Cleavage rates remained unchanged when 15 μM of NPS2143 was added to the culture medium (79.1 ± 6.8 vs. 73.7 ± 5.3; mean % ± SEM; p > 0.05, control vs. inhibitor). By contrast, development to the morula (46.6 ± 7.3 vs. 24.3 ± 4.3; mean % ± SEM; p < 0.05) and blastocyst stages (29.9 ± 9.0 vs. 9.9 ± 3.6; mean % ± SEM; p < 0.05) decreased (control vs. inhibitor). Our results demonstrate a key role of CaSR on sperm motility and embryo development but not on oocyte maturation or fertilization in the bovine species.

In vitro fertilization (IVF) is an established technology that is widely used in reproductive engineering. However, in rats, successful application of IVF is difficult to achieve, and it has had poor reproducibility. In a previous study on the critical issues associated with successful IVF in Wistar rats, we investigated the influence of oocyte collection duration on fertilization rates by dividing the procedure into three steps (oviduct extraction from euthanized animals, oocyte collection from the ampullae of oviducts, and oocyte preincubation until insemination), and identified the appropriate times for each. Here we show that use of the same defined duration for oviduct extraction from superovulated Wistar rats and for oocyte collection from the oviducts also produced highly reproducible fertilization rates of more than 90% in other rat strains. Furthermore, the versatility of these criteria was demonstrated using another IVF protocol. Thus, this simple procedure has enabled the standardization of IVF in rats and will enhance further experimental studies.

Although L-Arginine (ARG) has been reported as a promising bovine sperm capacitation agent, its effects on embryo development are still poorly understood. Herein, we compared the effects of ARG and/or heparin (HEP) addition to the fertilization medium for bovine oocytes on sperm capacitation and embryo development. We chose 10 mM ARG based on blastocyst development rates in a titration experiment. Addition of ARG and/or HEP to the fertilization medium resulted in similar rates of blastocyst development (P > 0.05). However, when ARG, but not HEP, was combined with a nitric oxide (NO) synthase inhibitor (N-Nitro-L-ARG-methyl ester, 10 mM) blastocyst development was decreased (P < 0.05). To assess the effects on capacitation, bovine sperm were incubated for 0, 3, and 6 hours in fertilization medium containing ARG and/or HEP and/or N-Nitro-L-ARG-methyl esterand acrosomal exocytosis rates were evaluated using fluorescein isothiocyanate conjugated Pisum sativum lectin (FITC-PSA) staining and flow cytometry. With HEP, acrosomal exocytosis rates were highest by 3 hours of incubation; however, by 6 hours, rates were similar for HEP and/or ARG (P > 0.05) and higher than those in control media (P < 0.05). Although both ARG and HEP increased sperm NO production (P < 0.05), combination with L-NAME only precluded acrosomal exocytosis when ARG added alone in the medium (P > 0.05). These results suggest that although both ARG and HEP supported sperm capacitation, only the effects of the former were driven via NO production. Moreover, ARG was also as effective as HEP at improving blastocyst development rates. Therefore, ARG may be used as a low-cost alternative sperm capacitation agent for bovine in vitro embryo production.

The purpose of this study was to investigate the effects of ram melatonin treatment on the sperm quality and metabolite composition of the seminal plasma in the non-breeding season. Four mature rams were treated with 54 mg melatonin in March subcutaneous implants and four untreated rams served as control. At 0, 30, 90 and 120 days semen samples were collected and sperm, separated from seminal plasma, was evaluated for its capacity to fertilize and produce embryos in vitro. Seminal plasma metabolites were extracted and analyzed by capillary electrophoresis/mass spectroscopy. In the resulting electropherograms, the area corresponding to selected metabolites was extracted and quantified. Ram melatonin treatment affected the in vitro fertilization competence of sperm. Blastocyst output increased until 90 days after treatment (27.20 ± 7.35 vs 54.7 ± 4.4% at 0 and 90 days respectively; p < 0.05) while the untreated group did not show statistical differences. In treated rams, the concentration of melatonin in seminal plasma increased from 3.34 ± 1.70 at day 0-9.65 ± 2.89 AU (Arbitrary Units) after 90 days, then decreased to reach the level of the untreated ram after 120 days (p < 0.05). During 90 days after melatonin treatment, an increase (p < 0.05) in seminal plasma concentrations of glutamic acid (6.28 ± 1.53 vs 14.93 ± 1.53 AU at 0 and 90 days respectively), glutamine (16.89 ± 4.65 vs 54.51 ± 4.65 AU), carnitine (22.97 ± 9.81 vs 104.30 ± 9.81 AU), acetyl-carnitine (48.15 ± 17.32 vs 217.69 ± 17.32 AU), choline (1.82 ± 1.55 vs 14.16 ± 1.55 AU) and arginine (1.31 ± 1.08 vs 14.25 ± 1.08 AU) was detected. Tyrosine concentration increased during 30 days from melatonin treatment (12.79 ± 3.93 vs 27.08 ± 3.04 AU) but at 90 days its levels were similar to the untreated group. In conclusion, melatonin treatment during the non-breeding season improves the concentration of several metabolites in seminal plasma and sperm fertilization competence in Sarda breed ram.

Prostacyclin (PGI2) is synthesised in oviductal fluid and enhance the embryo development during the preimplantation period. The objective of the present study was to determine the effect of an analogue of prostacyclin (iloprost) on the in vitro maturation (IVM) and the developmental competence of bovine oocytes. Cumulus oocyte complexes (COCs) were cultured in maturation medium with iloprost (0.5 μM) for 24 h. We found that iloprost assisted maturation rates and cumulus cell expansion of bovine oocytes, and it increased the mRNA expression of genes related to cumulus expansion: ADAM17, AREG, and TNFAIP6 and cathepsin genes (CTSK and CTSS). Moreover, iloprost reduced the occurrence of apoptosis in COCs and promoted an antiapoptotic balance in the transcription of genes involved in apoptosis (BAX and BCL2). COCs treatment with iloprost during IVM also reduced intracellular reactive oxygen species (ROS) levels, while glutathione (GSH) levels and the mRNA expression of antioxidant genes CAT and GPx4 were markedly increased. We also showed that an analogue of PGI2 influenced the mitochondrial status via distribution rates of mitochondria and mitochondrial membrane potential in oocytes. Although, iloprost-enhanced maturation had no direct effect on number of embryos cleaved, it increased blastocyst rates of bovine embryos as well as proportion of expanded blastocysts. These results indicate that the supplementation of maturation medium with iloprost is beneficial for the maturation efficiency and developmental competence of bovine oocytes.

Growth Differentiation Factor 8 (GDF8) is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator. This factor is expressed as a paracrine factor in mural granulosa cells. To investigate the effects of GDF8 on the in vitro maturation (IVM) of porcine oocytes, we assessed the quality of matured oocytes as well as the specific gene transcription and protein activation levels in oocytes and cumulus cells (CCs) after IVM and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). Supplemental concentrations (0, 1, 10, and 100 ng/ml) of GDF8 were provided in IVM medium. Supplementation with GDF8 during IVM induced transcription of specific TGF-β receptor genes, such as ActRIIb and Alk4/5, and the recognition of the GDF8 by these receptors induced phosphorylation of p38 MAPK. Activated p38 MAPK signaling changed oocyte maturation and cumulus expansion-related gene transcription: Nrf2 and Bcl-2 in oocytes and PCNA, Nrf2, Has2, Ptx3, and TNFAIP6 in CCs. The altered gene expression pattern during IVM resulted in a 10% lower level of intracellular ROS in mature oocytes. The improved cytoplasmic maturation led to an increase in the fertilization efficiency and subsequent embryonic developmental competence. The embryonic development showed increases in the blastocyst formation rate and higher transcription levels of POU5F1 and BCL-2 in the blastocysts. The present study suggests that supplementation of GDF8 during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by reducing the intracellular ROS level in oocytes by altering the transcription of specific genes and increasing the phosphorylation of p38 MAPK during IVM. In conclusion, for the first time, our results demonstrate that GDF8 can act as a paracrine factor to modulate oocyte maturation by regulating p38 MAPK phosphorylation and intracellular ROS level during porcine IVM.

The use of different sires influences in vitro embryo production (IVP) outcome. Paternal effects are observed from the first cleavages until after embryonic genome activation (EGA). Little is known about the mechanisms that promote in vitro fertility differences, even less about the consequences on embryo development. Therefore, this study aimed to evaluate the paternal effect at fertilization, embryo developmental kinetics, gene expression and quality from high and low in vitro fertility bulls. A retrospective analysis for bull selection was performed using the In vitro Brazil company database from 2012 to 2015. The dataset was edited employing cleavage and blastocyst rates ranking a total of 140 bulls. Subsequently, the dataset was restricted by embryo development rate (blastocyst/cleaved rate) and ten bulls were selected as high (HF; n = 5) and low (LF; n = 5) in vitro fertility groups. IVP embryos derived from high and low fertility bulls were classified according to their stage of development (2 cells, 3-4 cells, 6 cells, 8-16 cells), at 24, 36, 48, 60, 72 hpi, respectively, to evaluate embryo kinetics. Pronuclei formation (24 hpi), cleavage rate (Day 3), development rate, and blastocyst morphology (Grade I and II - Day 7) were also assessed, as well as the abundance of 96 transcripts at 8-16 cell stage and blastocysts. There was no difference in early embryo kinetics (P > 0.05), and cleavage rate (HF = 86.7%; LF = 84.9%; P = 0.25). Nevertheless, the fertilization rate was higher on HF (72%) than LF (62%) and the polyspermy rate was lower on HF compared to LF (HF:16.2% LF:29.2%). As expected, blastocyst rate (HF = 29.4%; LF = 16.0%; P < 0.0001) and development rate (HF = 33.9% LF = 18.9%; P < 0.0001) were higher in HF than LF. At the 8-16 cell stage, 22 transcripts were differentially represented (P ≤ 0.05) between the two groups. Only PGK1 and TFAM levels were higher in HF while transcripts related to stress (6/22, ∼27%), cell proliferation (6/22, ∼27%), lipid metabolism genes (5/22, ∼23%), and other cellular functions (5/22, ∼23%) were higher on LF embryos. Blastocysts had 9 differentially represented transcripts (P ≤ 0.05); being only ACSL3 and ELOV1 higher in the HF group. Lipid metabolism genes (3/9, 33%) and other cellular functions (6/9, 67%) were higher in the LF group. In conclusion, the timing of the first cleavages is not affected by in vitro bull fertility. However, low in vitro fertility bulls presented higher polyspermy rates and produced 8-16 cells embryos with higher levels of transcripts related to apoptosis and cell damage pathways compared to high in vitro fertility ones. Evidence such as polyspermy and increase in apoptotic and oxidative stress genes at the EGA stage suggest that embryo development is impaired in the LF group leading to the reduction of blastocyst rate.

This study aimed to assess the effects of carp pituitary extract (CPE), follicle stimulating hormone (FSH) and luteinizing hormone (LH) on zebrafish oocyte maturation and the ability of these mature oocytes to be fertilized and developed until hatching. Stage III follicles were matured in eight treatments: five concentrations of CPE (16, 32, 48, 64 and 80 μg/mL), one of FSH (0.5 μg/mL), one of LH (0.5 μg/mL), or one combination of FSH (0.5 μg/mL) and LH (0.5 μg/mL). Maturation rates in CPE treatments were 12.8% (16 μg/mL), 24.8% (32 μg/mL), 27.0% (48 μg/mL), 22.7% (64 μg/mL) and 9.6% (80 μg/mL); in FSH was 15.7% (0.5 μg/mL), in LH was 31.8% (0.5 μg/mL) and in FSH (0.5 μg/mL) combined with LH (0.5 μg/mL) it was 50.4%. In vitro fertilization was performed in all treatments; however, only the treatment combining FSH and LH resulted in fertilized oocytes. After maturation using FSH combined with LH, the cleavage rate was 33.3% and hatching rate of live larvae was 20.0%. These results showed that FSH combined with LH was effective in IVM of zebrafish oocyte.

Pregnancies obtained by Assisted Reproductive Technologies are at higher risk of miscarriage than those obtained naturally. Previously, we reported impaired placental vascular development of in vitro produced (IVP) sheep embryos and defective DNA methylation in the placentae of those embryos. One reason behind these observed defects may be an impaired One Carbon Metabolism (OCM) The present study was performed to test the hypothesis that Cobalamin (Vitamin B12, an important OCM co-factor) supplementation during IVM corrects DNA methylation of IVP embryos and, consequently, ameliorates placental vasculogenesis. To this aim, embryos derived from oocytes matured with Cobalamin (B12 group) or without (negative control group, -CTR) were transferred to synchronized recipient sheep. At day 20 of pregnancy, collected embryos were morphologically evaluated while placentae were subjected to qPCR and histological analysis. The positive control group (+CTR) consisted of conceptuses obtained from naturally mated sheep. Results showed an increased fertilization rate in the B12 group vs -CTR (69.56% vs 57.91% respectively, P = 0.006) not associated with quantitative improvement in blastocyst and/or implantation rate (44.32% vs 36.67% respectively, P > 0.05). Moreover, Cobalamin supplementation during oocyte IVM ameliorated resulting conceptuses quality, in terms of placental vascularization (vessels' maturity and vasculogenetic factors' expression). The expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was also improved in placentae from the B12 group. In conclusion, Cobalamin supplementation during oocyte IVM improves IVP embryo quality. These results suggest that Cobalamin should be included in standard IVM media.

In vitro fertility potential of individual bulls is still relatively uncharacterized. Classical sperm analysis does not include the evaluation of all sperm characteristics and thus, some cell compartments could be neglected. In humans, sperm DNA integrity has already proven to have major influence in embryo development and assisted reproduction techniques successfully. In bovine, some studies already correlated chromatin integrity with field fertility. However, none of those have attempted to relate DNA assessment approaches such as chromatin deficiency (CMA3), chromatin stability (SCSA; AO+) and DNA fragmentation (COMET assay) to predict in vitro bull fertility. To this purpose, we selected bulls with high and low in vitro fertility (n = 6/group), based on embryo development rate (blastocyst/cleavage rate). We then performed CMA3, SCSA test and COMET assay to verify if the difference of in vitro fertility may be related to DNA alterations evaluated by these assays. For the three tests performed, our results showed only differences in the percentage of cells with chromatin deficiency (CMA3+; high: 0.19 ± 0.03 vs low: 0.04 ± 0.04; p = 0.03). No difference for chromatin stability and any of COMET assay categories (grade I to grade IV) was observed between high and low in vitro fertility bulls. A positive correlation between AO + cells and grade IV cells was found. Despite the difference between groups in CMA3 analysis, our results suggest that protamine deficiency in bovine spermatozoa may not have a strong biological impact to explain the difference of in vitro fertility between the bulls used in this study.

Experiments were conducted in wood bison to determine the effect of additional maturation time on embryo development of in vivo matured oocytes. In experiment 1, cumulus-oocyte complexes (COC) were collected 30 hours after hCG treatment in superstimulated wood bison, and expanded COC were fertilized immediately or after 4 hours of additional in vitro maturation. Embryo development was assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). No difference in cleavage rate was detected (55.3% vs. 60.5%, P = 0.82), but the Day 8 blastocyst rate was higher after an additional 4 hours of in vitro maturation time (44.7 vs. 18.4%, P = 0.03). In experiment 2, COC were collected at either 30 hours or 34 hours after hCG treatment. Expanded COC from the 30 hours group were fertilized after 4 hours of in vitro maturation, whereas those from the 34 hours group were fertilized immediately. A higher cleavage rate (74.3 vs. 57.0%) and blastocyst rate (54.1 vs. 37.2%) were found in the 34 hours group versus the 30 hours group (P < 0.05). In conclusion, an additional short period of in vitro maturation, or an extended period of in vivo maturation are beneficial for in vitro embryo production in wood bison.

Assisted reproductive techniques (ART) have been widely used in farm animals in the last decades. Sexed cryopreserved spermatozoa, ovum pick up, in vitro embryo production and transfer constitute the ART that have revolutionized the dairy industry. However, the efficiency of some of these techniques is still low due in part to sperm quality, which influences fertilization, embryo development and implantation. The Sperm Selection Assay (SSA), based on sperm chemotaxis towards progesterone, provides a sperm subpopulation enriched with spermatozoa that are capacitated, with intact DNA and low level of oxidative stress. Since the SSA selects a sperm subpopulation at optimum physiological state, the application of the SSA may improve the efficiency of the current ART. The aim of this study was to adapt the SSA for unsexed and sexed bovine frozen-thawed semen samples, and then to test whether sperm selection by the SSA improves the cleavage rate of bovine embryos in vitro. The optimal SSA conditions to obtain the higher sperm accumulation percentage given by chemotaxis were the same for both unsexed and sexed semen samples. Thus, sperm accumulation in W2 was significantly higher when: 2 million sperm per mL were placed in W1 (unsexed samples: 12 ± 1%, p = 0.002; sexed samples: 14 ± 3%, p = 0.02); 1 pM progesterone was placed in W2 (unsexed sample: 9 ± 1%, p = 0.009; sexed samples: 11 ± 2%, p = 0.02); and to incubate the SSA device for 10 min (unsexed samples: 17 ± 2%, p = 0.007; sexed samples: 10 ± 1%, p = 0.004). We found that the quality of spermatozoa recovered from W2 in unsexed and sexed semen was enhanced. Thus, the capacitation index was significantly increased (unsexed samples: 1.75 ± 0.1, p = 0.0001; sexed samples: 1.76 ± 0.2, p = 0.004), while DNA fragmentation index was significantly decreased (unsexed samples: 0.33 ± 0.07, p = 0.0003; sexed samples: 0.32 ± 0.04, p = 0.002). Moreover, the cleavage index of oocytes fertilized with either unsexed or sexed SSA-selected sperm was significantly improved (unsexed samples: 3.2 ± 0.4, p = 0.0001; sexed samples: 2.3 ± 0.33, p = 0.03). Thus, we show that the SSA can be used to recruit a bovine sperm subpopulation at optimal functional state regardless of whether the sample is previously sexed, and that this optimal state improves bovine embryo cleavage rate.

In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are important breeding techniques for livestock. High-quality MII oocytes produced from in vitro maturation (IVM) are required for the two techniques listed above. The ovaries used for IVM operations are primarily acquired from commercial abattoirs, and the pathogen status of slaughtered animals becomes an unavoidable issue. Our previous monitoring data showed that porcine circovirus type 2 (PCV-2) is the main pathogen present in ovaries from abattoirs. However, the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production (IVP) of embryos are unclear, and currently there are no relevant studies. Therefore, the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development. More than five hundred ovaries and five thousand oocytes were utilized in the present study. Polymerase chain reaction (PCR) was used to detect PCV-2 DNA in ovaries, follicular fluid (FF), oocytes, cumulus cells and IVP embryos. The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated. We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR. Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a. PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation. Moreover, virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers. Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium. For the first time, this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.

The present study was designed to define the impact of l-carnitine, supplemented during maturation, on bovine oocytes with different meiotic competence in terms of their IVF outcomes. Meiotically more competent (MMC) and less competent (MLC) oocytes were obtained separately from differently sized follicles at selected phases of folliculogenesis. The oocytes were matured with or without l-carnitine, fertilized and cultured to the blastocyst stage. The oocytes were examined for nuclear maturation, mitochondrial cluster formation, lipid consumption, fertilization and embryo development. The proportion of oocytes at metaphase II was significantly higher in the l-carnitine-treated MMC than that in the l-carnitine-treated MLC oocytes. However in comparison with the untreated controls, the proportion of MII oocytes with mitochondrial clusters was significantly higher only in the l-carnitine-treated MLC oocytes, which also showed a significantly lower mean lipid content. The l-carnitine-treated MLC oocytes showed significantly higher fertilization and syngamy rates than the untreated MLC oocytes. On the other hand, in the l-carnitine-treated MMC oocytes, the fertilization rate was similar to that of the untreated controls and the syngamy rate was significantly delayed. Although no significant differences in cleavage on Day 2 were found among all oocyte categories, l-carnitine treatment resulted in a significantly higher blastocyst yield in the MLC oocytes on Day 7 and Day 8 and a significantly higher proportion of expanded blastocysts in relation to the total number of blastocysts in MMC oocytes on Day 8. It can be concluded that l-carnitine supplementation during maturation improves the development of bovine embryos from meiotically less competent oocytes and accelerates blastocyst formation from more competent oocytes.

The supplementation of dimethyl alpha-ketoglutarate (DMKG) during the in vitro maturation (IVM) process has been shown to improve the in vitro developmental competences of porcine oocytes. Here, the effects of DMKG supplementation in IVM medium on the development competencies of ovine oocytes were investigated by analyzing the nuclear maturation rate to metaphase II (MII) stage, ATP synthesis, cortical granules (CGs) dynamic, F-actin polymerization, mitochondrial activity, mitochondrial damage, reactive oxygen species (ROS) production, intracellular glutathione (GSH) production, DNA damage, cellular apoptosis, fertilization capacity and blastocyst development potential of ovine oocytes. In addition, the oxidative stress damage model induced by H2O2 treatment was applied to confirm the antioxidative effect of DMKG supplementation on the development of ovine oocytes. The results showed that compared with MII oocytes without DMKG supplementation (Control group), 3 mM DMKG supplementation during IVM significantly (P < 0.05) increased nuclear maturation rate, ATP synthesis, CGs dynamic, F-actin polymerization, mitochondrial activity, GSH production and embryonic developmental competence and decreased ROS production, mitochondrial damage, DNA damage and cellular apoptosis level of ovine MII oocytes. Moreover, the reductions in the developmental competences of ovine MII oocytes caused by H2O2 induced oxidative stress damages were effectively ameliorated by the co-supplementation in IVM of 3 mM DMKG (P < 0.05). Our results demonstrate the promising effect of DMKG supplementation on the in vitro developmental competence of ovine oocytes via the reduction of oxidative stress damages and indicates further research into the clinical applications of DMKG and the development of ovine breeding technologies is warranted.

Two experiments were done to test the hypothesis that morphologic characteristics of wood bison cumulus-oocyte complexes (COC) are reflective of the ability of the oocyte to develop to an advanced embryonic stage after in vitro maturation, fertilization and culture, and to determine the effect of prolonging the interval from the end of superstimulation treatment to oocyte collection (FSH starvation period). Experiments were done during the anovulatory season. In Experiment 1, ovarian superstimulation was induced in 10 bison with two doses of FSH given at 48 h intervals beginning at the time of follicular wave emergence. COC were collected 3 days (72 h) after the last dose of FSH by follicular aspiration and classified as compact, expanded or denuded. The COC were matured in vitro for 24 h before fertilization in vitro (Day 0). Embryo development was assessed on Days 3, 7 and 8. The blastocyst rate was 7/34, 2/10 and 0/3 in COC classified as compact, expanded and denuded, respectively; however, only compact COC resulted in embryos that reached the expanded blastocyst stage. In Experiment 2, COC were collected at either 3 or 4 days (72 or 96 h) after the last dose of FSH (n = 16 bison/group) to determine the effect of the duration of FSH starvation on oocyte competence. The COC were classified as compact good (>3 layers of cumulus cells), compact regular (1-3 layers of cumulus cells), expanded or denuded, and then matured, fertilized and cultured in vitro. Although follicles were larger (P < 0.05) in the 4-day FSH starvation group, there was no effect of starvation period on the distribution of COC morphology; overall, 112/194 (57.7%) were compact, 29/194 (26.3%) were expanded, 39/194 (20.1%) were denuded, and 14/194 (7.2%) were degenerated (P < 0.05). Similarly, there was no effect of starvation period on embryo development. Compact good COC had the highest cleavage (88%) and blastocyst rates (54%; P < 0.05), followed by compact regular COC at 73% and 25%, respectively. Expanded and denuded COC had low cleavage (40% vs. 59%, respectively) and blastocyst rates (5% vs. 8%, respectively). We conclude that morphologic characteristics of wood bison COC are reflective of the ability of the oocyte to develop into an embryo in vitro. Importantly, oocytes collected from superstimulated bison during the anovulatory season were competent to develop to the blastocyst stage following in vitro maturation, fertilization and culture.

Oocyte maturation in culture is still the weakest part of in vitro fertilization (IVF) and coculture with somatic cells may be an alternative to improve suboptimal culture conditions, especially in the pig in which maturation takes more than 44 h. In the present study, we investigated the effect of a coculture system of porcine luteal cells (PLC) during in vitro maturation (IVM) on embryo development and gene expression. Cumulus-oocyte complexes were matured in vitro in TCM-199 with human menopausal gonadotrophin (control) and in coculture with PLC. IVF was performed with frozen-thawed boar semen in Tris-buffered medium. Presumptive zygotes were cultured in PZM for 7 days. The coculture with PLC significantly increased blastocysts rates. Gene expression changes were measured with a porcine embryo-specific microarray and confirmed by RT-qPCR. The global transcription pattern of embryos developing after PLC coculture exhibited overall downregulation of gene expression. Following global gene expression pattern analysis, genes associated with lipid metabolism, mitochondrial function, endoplasmic reticulum stress, and apoptosis were found downregulated, and genes associated with cell cycle and proliferation were found upregulated in the PLC coculture. Canonical pathway analysis by Ingenuity Pathway revealed that differential expression transcripts were associated with the sirtuin signaling pathway, oxidative phosphorylation pathway, cytokines and ephrin receptor signaling. To conclude, the coculture system of PLC during IVM has a lasting effect on the embryo until the blastocyst stage, modifying gene expression, with a positive effect on embryo development. Our model could be an alternative to replace the conventional maturation medium with gonadotrophins with higher rates of embryo development, a key issue in porcine in vitro embryo production.

The bovine oviduct provides the site for fertilization and early embryonic development. Modifications to this physiological environment, for instance the presence of pathogenic bacterial species, could diminish reproductive success at early stages of pregnancy. The aim of this study was to elucidate the inflammatory responses of bovine oviductal epithelial cells (BOEC) to a pathogenic bacterial species (Trueperella pyogenes) and a potentially pathogenic bacterium (Bacillus pumilus). BOEC from four healthy animals were isolated, cultured in passage 0 (P0) and passaged until P3. Trypan blue staining determined BOEC viability during 24 h co-culture with different multiplicities of infection (MOI) of T. pyogenes (MOI 0.01, 0.05, 0.1 and 1) or B. pumilus (MOI 1 and 10). BOEC remained viable when co-cultured with T. pyogenes at MOI 0.01 and with B. pumilus at MOI 1 and 10. Extracted total RNA from control and bacteria co-cultured samples was subjected to reverse transcription-quantitative polymerase chain reaction (RTq-PCR) to determine mRNA expression of various studied genes. The rate of release of interleukin 8 (IL8) and prostaglandin E2 (PGE2) from BOEC was measured by ELISA after 24 h co-culture with bacteria. RT-qPCR of various selected pro-inflammatory factors revealed similar mRNA expression of pro-inflammatory factors in BOEC co-cultured with T. pyogenes and in the controls. Higher mRNA expression of IL 1A, -1B, tumor necrosis factor alpha and CXC ligand (CXCL) 1/2, -3, -5 and IL8 and PG synthesis enzymes in BOEC co-cultured with B. pumilus was observed. In the presence of B. pumilus a higher amount of IL8 and PGE2 was released from BOEC than from controls. The viability and pro-inflammatory response of P3 BOEC incubated with bacteria was lower than in P0 BOEC. These findings illustrate the pathogenicity of T. pyogenes towards BOEC in detail and the potential role of B. pumilus in generating inflammation in oviductal cells. Culturing conditions influenced the pro-inflammatory responses of BOEC towards bacteria. Therefore, researchers conducting epithelial-bacterial in vitro co-culture should not underestimate the effects of these parameters.

Expression of myostatin (MSTN, also known as growth differentiation factor 8, GDF8) was recently detected in cumulus-oocytes complexes (COCs), however little is known about its role in in vitro maturation (IVM) and fertilization (IVF) in large animals. Therefore, this study was designed to investigate the effect of MSTN inhibition on IVM of buffalo oocytes through investigation of IVM efficiency and expression of some specific genes in COCs from IVM till subsequent developmental stages following IVF. To reach this goal, we prepared a construct of adeno-associated virus (AAV) carrying MSTN pro-peptides (AAV-MSTNP) to inhibit MSTN. Over-expression of MSTNP was verified by upregulated expression of MSTNP and downregulated expression of the TGFβ receptor ActRIIb, the TGFβ signal transducer SMAD2 in COCs using qPCR. Microinjection of AAV-MSTNP to oocytes before IVM yielded a significant decrease in maturation rate as revealed by less cumulus cells expansion, fewer oocytes reaching metaphase II, and downregulation of cumulus expansion-related genes pentraxin 3 (Ptx3) and prostaglandin-endoperoxide synthase 2 (Ptgs2) as compared to the control and vehicle groups. These changes were also accompanied by elevated intracellular reactive oxygen species (ROS), upregulated expression of the apoptotic Bax gene, reduced antioxidant enzymes (SOD, CAT, GPX) activities, and downregulated expression of the antioxidant gene nuclear factor erythroid 2 like 2 (Nrf2), and the anti-apoptotic gene Bcl2 in COCs after IVM. Overexpression of MSTN inhibitor, MSTNP, also inhibited GDF9 and BMP15 genes expression in COCs. Additionally, both the fertilization efficiency and cleavage and blastocyst rates were significantly lower in MSTNP group than in the control and vehicle groups. The obtained data suggest an important role for MSTN during IVM and the subsequent developmental stages probably through, at least in part, inhibition of ROS production and apoptosis and modulation of IVM-related gene expression in COCs.

Successful cryopreservation of bison semen is fundamental for restoration of genetic diversity in Canada's wood bison. Conventional bovine semen extenders contain animal products, such as egg yolk and milk, which are undesirable because of biosecurity risks and undefined composition. In this study, we examined the efficacy of an exogenous protein-free extender containing cholesterol-cyclodextrin complex (CC) to cryopreserve bison semen. The study also provided an opportunity to determine the effectiveness of different ovulation synchronization protocols for fixed-time artificial insemination in bison. Semen was collected from wood bison bulls via electroejaculation and cryopreserved in either Tris-egg yolk-glycerol (called 'TEYG') extender or pretreated with CC (2 mg/mL semen) and diluted in Tris-glycerol (collectively called 'CC-TG') extender. Post-thaw sperm motion characteristics and in vitro fertilization of cattle oocytes confirmed that CC alone without egg yolk protected bison sperm during cryopreservation process. In the first fertility trial, however, no pregnancy was obtained following fixed-time artificial insemination of bison cows with CC-TG extender. In a follow-up trial, low concentration of CC (1 mg/mL semen) resulted in better post-thaw sperm motion characteristics, fertility rate, and birth of live calves following fixed-time artificial insemination. Results showed that 1 mg CC/mL semen completely replaced egg yolk in bison semen extender. In addition, both follicular ablation and steroid treatment protocols provided ovulation synchrony to permit successful application of fixed-time artificial insemination in bison. This is the first report on the birth of live bison calves following fixed-time artificial insemination using semen cryopreserved in a defined extender.