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Sex preselection has always generated great interest among livestock producers. Among the prevalent sperm sorting methods, there is much evidence that sex sorting has a negative effect on sperm quality with an altered pattern of sperm motility, ultimately reducing the period of cell viability. In this study, we have established a new approach for the preselected embryo production by using WholeMom®; a monoclonal antibody developed against bull sperm epitopes for simple and easy separation of X- and Y-sperm. There were no significant differences (P > 0.05) in the percentage of presumptive zygotes between the control and the X-sperm sorted group, but there was a difference in early cleaving embryos with there being 81.2 ± 1.4%, 78.3 ± 1.0%, and 66.7 ± 1.1% for the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Similarly, the percentage of embryos that developed to the blastocyst stage (Day 7) were also greater (P < 0.05) in the control and X-sperm sorted group compared with the Y-sperm sorted group being 34.8 ± 1.0%, 32.1 ± 0.8%, and 23.7 ± 1.0% in the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Furthermore, B- SRY F2 and B- SRY R2 gene expression data indicated there was a detection accuracy of 81.0% for the female embryos and 72.5% for the male embryos produced in vitro. In conclusion, in cattle in vitro derived embryo production using pre-selected sexed semen and subsequent embryo transfer can facilitate the mass production of individuals that are genetically superior. [ABSTRACT FROM AUTHOR]

Highlights • Foaling augmentation may shorten the interval between foaling and standing. • Foaling augmentation may increase the interval between foaling and nursing. • Foaling augmentation does not increase risk of retained fetal membranes. Abstract Augmentation of parturition can be used to advance labor in mares to occur at a time when personnel is available to assist if necessary. We performed a retrospective study to determine the efficacy and safety of augmentation to manage foalings. Augmentation was performed with 3 IU oxytocin i.v. when mammary calcium concentrations were ≥250 ppm, mammary secretion pH ≤ 6.5, and the mare showed impending signs of parturition. Augmented parturitions (n = 19) were compared with three different control groups. The three control groups were: 1) Time Match control (n = 37) which were non-augmented foalings in the barn during the same time period; 2) Mare Match control (n = 32) which were the non-augmented parturitions of the augmented mares in previous years; and 3) Historic Match control (n = 165) consisted of foalings that occurred from 2006 to 2016 in the facility. All augmented mares foaled within two h with an average of 44 min (range 20–75) after oxytocin injection. The interval between foaling and the foal standing was shorter in augmented parturitions compared with historic match controls. The interval between foaling and the foal nursing was longer with augmented parturitions compared with time match and historic match controls. Duration of fetal membrane retention was not different between all groups. Augmentation of imminent parturition is potentially a safe and effective treatment for mares and foals. Implementation of augmentation as a routine procedure may increase the likelihood of enteral administration of colostrum to foals. [ABSTRACT FROM AUTHOR]

The aim of the present study was to compare differences in composition between in vitro cultured early developmental embryos resulting from either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Non-invasive metabolomic profiling of culture media was conducted with laser tweezer Raman spectroscopy (LTRS), providing molecular information that was used to aid the diagnosis or treatment of embryos that were adversely affected by ICSI treatment, ultimately improving the ICSI embryonic developmental potential. Cattle embryos were generated via ICSI and IVF with development to the 2-, 4-, 8-, 16-,32-cell, and blastocyst stages with individual in vitro culturing occurring for 4 h. The culture media for embryos in different developmental stages were separately analyzed using LTRS. The resulting composition of culture media used for culturing IVF- and ICSI-derived embryos was mainly altered in contents of carbohydrates, lipids, DNA, and proteins. Bands at 1004 cm −1 (phenylalanine) and 1529 cm −1 (-C = C-carotenoid) had specific patterns related to the metabolicactivity of embryos; using LTRS, and these may be considered as biomarkers for embryonic development. Furthermore, the vibrations of lipids at different stages increased more with assessment of ICSI culture media than in IVF media. Discriminant function analysis can be utilized for the classification of culture media used for culture of ICSI- and IVF-derived embryos. In conclusion, LTRS can be used for development of an independent assay to assess embryo status during both ICSI and IVF procedures, which provides novel insights into differences in structure and components of single cells. [ABSTRACT FROM AUTHOR]

The ICSI-sperm mediated gene transfer (ICSI-SMGT) has been used to produce transgenic mice with high efficiency; however, the efficiency of this technique in farm animals is still less than desirable. Pretreatment of sperm with membrane destabilizing agents can improve the efficiency of ICSI in cattle. The objective of the present study was to evaluate streptolysin-O (SLO) as a novel treatment to permeabilize the bovine sperm membrane and assess its effect on efficiency of generating transgenic embryos by ICSI-SMGT. First, there was evaluation of the plasma membrane integrity (SYBR/PI), acrosome membrane integrity (PNA/FITC), DNA damage (TUNEL) and binding capacity of exogenous DNA (Nick Translation) in bull sperm treated with SLO. Subsequently, there was assessment of embryonic development and the efficiency in generating transgenic embryos with enhanced expression of the gene for green fluorescent protein (EGFP). Results indicate that SLO efficiently permeabilizes the plasma and acrosome membranes of bull spermatozoa and increases binding of exogenous DNA mostly to the post-acrosomal region and tail without greatly affecting the integrity of the DNA. Furthermore, treatment of bull spermatozoa with SLO prior to the injection of oocytes by ICSI-SMGT significantly increased the rate of embryo expression of the EGFP gene. Future experiments are still needed to determine the effect of this treatment on the development and transgene expression in fetuses and animals produced by ICSI-SMGT. [ABSTRACT FROM AUTHOR]

Endogenous peroxiredoxin II (PRDX II) protein plays a vital role in early embryonic development. This study assessed the beneficial effects of exogenous PRDX II on bovine embryo development at the cellular and molecular levels. To this end, in vitro maturation (IVM) medium was supplemented with various concentrations of PRDX II (0, 6.25, 12.5, 25, 50, and 100 μg/mL). Of these, 12.5 μg/mL PRDX II was the most effective and significantly promoted embryonic development. Therefore, this concentration of PRDX II was used in subsequent experiments. The percentage of embryos that developed into Day 8 blastocysts and the total number of cells per blastocyst (38.2% and 150.6 ± 5.1) was higher in the PRDX II-treated group than in the control (26.4% and 128.9 ± 3.9, respectively). Moreover, the percent of TUNEL positive cells was higher ( P < 0.05) in the control than in the PRDX II-treated. Furthermore, PRDX II added to the IVM media increased mitochondria content in blastocysts and decreased the intracellular ROS levels in oocytes and blastocysts compared with the control ( P < 0.05). The expression of genes associated with blastocyst quality ( CDX2 and IFNτ ), antioxidant activity (SOD2), and mitochondrial activity (TFAM) was higher, whereas the expression of a gene involved in the apoptotic pathway ( c-FOS ) was lower, in the PRDX II-treated than in the control group. In conclusion, supplementation of IVM medium with PRDX II promotes development to the blastocyst stage and improves blastocyst quality through reducing ROS, enhancing embryonic mitochondrial activity, and modulating development- related target genes expression. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate whether resveratrol supplementation of bovine culture medium improves in vitro blastocyst development, embryo cryotolerance and cell numbers. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, zygotes were cultured in SOF medium, supplemented with 0 (control, n = 439), 0.25 μM ( n = 422), 0.5 μM ( n = 447) and 1 μM resveratrol ( n = 416). On Day 7 (IVF = Day 0) blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide and 0.5 M sucrose. Development rate, i.e. the percentage of embryos resuming development to reach a more advanced stage, and hatching rate were evaluated after 24 and 48 h culture. Blastocysts cultured with (0.5 μM) and without resveratrol underwent differential staining to count inner cell mass (ICM) and trophectoderm (TE) cells. Resveratrol during culture did not increase blastocyst yields (57.1, 57.7, 59.2 and 46.6%, respectively in 0, 0.25, 0.5 and 1 μM resveratrol). However, 0.5 μM resveratrol improved embryo cryotolerance compared to the control, as indicated by higher development rates (67.3% vs 50.3%, respectively; P < 0.01) and hatching rates (58.9% vs 30.9%, respectively; P < 0.01) recorded after 48 h post-warming culture. Blastocysts produced in the control and in 0.5 μM resveratrol groups had similar numbers of ICM (34.1 and 36.4, respectively), TE (88.1 and 85.3, respectively) and total (122.2 and 121.7, respectively) cells. In conclusion, low levels of resveratrol during in vitro culture improve the quality of IVP bovine embryos, as indicated by their increased resistance to cryopreservation. [ABSTRACT FROM AUTHOR]

Abstract: This experiment was conducted to evaluate endometrial echotexture in oestrus and early pregnancy and its association with ovarian hormones and foetal count in goats. Akkeci goats (Saanen×Kilis crossbreed, n =40) were randomly divided into two groups. Ten does (NAT) were mated on natural oestrus and 30 does (SYN) were subjected to synchronisationprior to mating. The uterus was scanned on the days of sponge insertion (d −14), sponge removal (d −2) and mating (d 0) as well as 17 (d 17) and 30 (d 30) days after mating. Mean gray level (MGL), homogeneity (HOM) and contrast (CON) values were calculated. Blood samples were collected on days ultrasonography was performed. Data were analyzed by Chi-square, ANOVA, regression tests. HOM value reached the highest level on the mating day and then continuously decreased (P <0.0001). Overall, HOM values were greater for SYN does than for NAT does after mating. CON values were virtually stable during the experimental period. MGL value fluctuated during the breeding period (P <0.03) at a similar fashion in NAT and SYN does. Foetal count was not correlated with plasma hormones and echotexture parameters. Plasma progesterone concentration was correlated with echotexture parameters (r =−0.28 for HOM; r =0.29 for CON; r =0.25 for MGL; P <0.05 for all) during post-mating. In conclusion, echotexture parameters changed during the breeding period, in association with plasma progesterone concentration. Future studies should test if the echotextural changes during embryonic fixation days can be used as a marker for early detection of pregnancy in does. [Copyright &y& Elsevier]

Abstract: Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10μM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P <0.05). A third experiment (experiment C) was performed to quantify and localise ADRP protein. Boec, in vivo and control groups were tested. After immunofluorescence staining followed by confocal microscopy analysis, embryonic ADRP was clearly localised around lipid droplets, indicating that ADRP is also a lipid droplet coat protein in bovine embryos. In conclusion, our results demonstrate that bovine embryos at the blastocyst stage expressed ADRP mRNA and protein, and that the embryonic culture system modified this expression. [Copyright &y& Elsevier]

Abstract: Human bactericidal/permeability-increasing protein (hBPI) is the only antibacterial peptide which acts against both gram-negative bacteria and neutralizes endotoxins in human polymorphonuclear neutrophils; therefore, hBPI is of great value in clinical applications. In the study, we constructed a hBPI expression vector (pBC1-Loxp-Neo-Loxp-hBPI) containing the full-length hBPI coding sequence which could be specifically expressed in the mammary gland. To validate the function of the vector, in vitro cultured C127 (mouse mammary Carcinoma Cells) were transfected with the vector, and the transgenic cell clones were selected to express hBPI by hormone induction. The mRNA and protein expression of hBPI showed that the constructed vector was effective and suitable for future application in producing mammary gland bioreactor. Then, female and male goat fibroblasts were transfected with the vector, and two male and two female transgenic clonal cell lines were obtained. Using the transgenic cell lines as nuclear donors for somatic cell nuclear transfer, the reconstructed goat embryos produced from all four clones could develop to blastocysts in vitro. In conclusion, we constructed and validated an efficient mammary gland-specific hBPI expression vector, pBC1-Loxp-Neo-Loxp-hBPI, and transgenic hBPI goat embryos were successfully produced, laying foundations for future production of recombinant hBPI in goat mammary gland. [Copyright &y& Elsevier]

Abstract: The study assesses the possibility to estimate the potential fertility of post-thawed antelope (Antidorcas marsupialis), impala (Aepyceros melampus) and blesbok (Damaliscus dorcus phillipsi) epididymal sperm using homologous and heterologous IVF and the functioning of cattle IVF system to produce antelope embryos. Cauda epididymal sperm were collected from the antelope and cryopreserved under field conditions. In vitro matured domestic cow, blesbok and springbok oocytes were co-incubated in modified-Tyrode Lactate (m-TL) IVF media with springbok, impala and blesbok sperm for heterologous IVF and springbok and blesbok sperm for homologous IVF. A group of presumptive zygotes from each treatment were examined for sperm penetration and male pronuclear formation after 18h and the remainder were cultured and evaluated for embryo cleavage 22h later. The study shows that Modified Tyrode Lactate in vitro fertilization media supports survivability, capacitation and hyperactivation of springbok, impala and blesbok sperm. Springbok, impala and blesbok post-thawed epididymal spermatozoa are capable of fertilizing domestic cow oocytes under conditions that support domestic cattle IVF. Penetration, male pronuclear formation and embryo cleavage did not differ (p >0.05) between cow oocytes inseminated with sperm from springbok, impala or blesbok however these parameters were higher (p <0.05) for oocytes inseminated with bull sperm. Modified Tyrode Lactate IVF media supported homologous fertilization and embryo development in springbok and blesbok however did not support blastocyst development. These findings suggest that cattle provide a useful model for evaluating springbok, impala and blesbok post-thawed cauda epididymal sperm functionality. Domestic cattle embryo culture conditions need to be modified to promote blastosyst development in these antelope species. Such research provides an important tool in assisted reproductive technology development when high biological value material is utilized for wild species recovery plans. [Copyright &y& Elsevier]

Abstract: The insulin-like growth factor (IGF) system is related to quality of oocytes and embryos. The aim of this study was to investigate the mRNA levels of IGF1 and IGF2 and their receptors, IGFR1 and IGFR2, as well as IGFBP2, IGFBP4, and PAPP-A in oocytes from Nelore compared to Holstein cows. Pools of oocytes (20 oocytes/pool) from Nelore (n =8 pools) and Holstein (n =4 pools) were obtained via ovum pick-up (OPU, 10 sessions) and cumulus cells and zona pellucida were removed. The pools were submitted to total RNA extraction. Expression of members of the IGF system was assessed by real time RT-PCR. The mRNA expression of IGF1 and IGF2, IGFR1 and IGFR2, IGFBP2 and IGFBP4 was significantly higher (P <0.01) in oocytes from Holstein whereas the expression of PAPP-A was significantly higher (P <0.05) in oocytes from Nelore cows. The high PAPP-A expression and the low expression of IGFBP2 and IGFBP4 are associated with more efficient degradation of IGFBPs, which results in greater bioavailability of IGF in Nelore oocytes when compared to the Holstein. [Copyright &y& Elsevier]

Abstract: Effects of enucleation timing on enucleation rates, development and methylation levels of reconstructed bovine embryos were investigated. However, the enucleation rate of reconstructed embryos produced by the enucleation before fusion and activation (EBFA) was higher than that by the enucleation after fusion and activation (EAFA) procedure (80.7% vs. 59.1%, P <0.05). The blastocyst rate of reconstructed embryos cloned with ear fibroblasts in EBFA group was reduced (P <0.05) in comparison with that of EAFA group (24.6% vs. 34.4%). Two out of 11 recipients were pregnant and gave birth to two viable calves after transfer of 20 reconstructed EBFA embryos. Two out of seven recipients were pregnant and also gave birth to two calves, with one surviving, after transfer of 12 reconstructed embryos produced by EAFA procedure. Finally, the methylation level of satellite I gene of donor cells (69.8%) and reconstructed embryos in EBFA group (64.7%) were similar, which were both higher (P <0.05) than that of the reconstructed embryos in EAFA group (44.4%). The methylation level of satellite I gene of the reconstructed embryos in the IVF embryos (31.9%) was lower (P <0.05) than those in all other treatments. In conclusion, the reconstructed bovine embryos produced by the EAFA procedure revealed a better developmental competence with a lower methylation rate of satellite I gene than those produced by the EBFA procedure. [Copyright &y& Elsevier]

Abstract: Oocyte cytoplasm plays a prominent role in cloned embryonic development. To investigate the influence of oocyte cytoplasmic amount on cloned embryo development, we generated bovine somatic cell nuclear transfer (SCNT) embryos containing high (30–40% of the cytoplasm was removed), medium (15–25% of the cytoplasm was removed) and low (<10% of the cytoplasm was removed) nucleocytoplasmic volume ratios (N/C) using enucleated metaphase II oocyte as recipient, and fibroblast as donor nucleus, and analyzed the expression levels of ND1, Cytb and ATPase6, as well as the embryonic quality. The results indicated: (1) the process of embryonic development was not influenced by <40% of cytoplasm removal; (2) the rate of blastocyst formation, the total number of blastomere and the ratio of ICM to TE were inversely proportional to the N/C; (3) SCNT embryos with reduced volume equal to 75–85% or >90% of an intact oocyte volume showed similar karyotype structure of the donor cells; (4) the number of mtDNA copy was larger in low N/C embryos than that in medium or high N/C embryos, and the expression levels of each gene hardly varied from the 2-cell to 8-cell stage, while the expression levels increased dramatically at the blastocyst stage; (5) from 16-cell to the blastocyst stage, the change of the expression level of each gene was not significant between low N/C embryos and IVF embryos, but it was more significant than those of high or medium N/C embryos. The results suggest that the decrease of mtDNA copy number and mitochondrial gene expression may be related to the impairment in early embryonic development, and removal of <10% adjacent cytoplasm volume may be optimal for bovine SCNT embryo development. [Copyright &y& Elsevier]

Abstract: Several studies using transrectal ovarian ultrasonic scanning in Bos taurus (B. taurus) cattle and more recently in Bos indicus (B. Indicus) females evaluated the reproductive cycles of heifers and cows under different conditions. In general, B. indicus cattle have more follicles and more follicular waves during the estrous cycle and ovulate from smaller follicles than B. taurus. Consequently B. indicus females have smaller corpora lutea and it is assumed circulating concentrations of estradiol and progesterone are also less. However, these findings may vary depending on the nutritional status and regimen in which the animals are managed. Moreover, there are significant differences between B. taurus and B. indicus regarding follicle size at the time of deviation of the dominant follicle. These differences in ovarian function between B. indicus and B. taurus, e.g. greater antral follicle population are, probably, the main reasons for the great success of in vitro embryo production programs in Zebu cattle, especially in Brazil. [Copyright &y& Elsevier]

Abstract: The possibility of producing interspecies handmade cloned (iHMC) embryos by nuclear transfer from donor cells of cattle, goat and rat using buffalo oocytes as recipient cytoplasts was explored. Zona-free buffalo oocytes were enucleated by protrusion cone-guided bisection with a microblade. After electrofusion with somatic cells, reconstructed oocytes were activated by calcimycin A23187, treated with 6-dimethylaminopurine and were cultured in K-RVCL-50® medium for 8 days. Although the cleavage rate was not significantly different when buffalo, cattle, goat or rat cells were used as donor nuclei (74.6±3.8, 82.8±5.3, 86.0±4.9 and 82.3±3.6%, respectively), the blastocyst rate was significantly higher (P <0.01) for buffalo (51.4±2.6) than for cattle (3.5±1.0) or the goat (2.2±0.9), whereas none of the embryos crossed the 32-cell stage when rat cells were used. However, the total cell number was similar for buffalo–buffalo (175.0±5.07) and cattle–buffalo embryos (178.0±11.84). Following transfer of 3 buffalo–buffalo embryos each to 6 recipients, 3 were found to be pregnant, though the pregnancies were not carried to full term. These results suggest that interspecies blastocyst stage embryos can be produced by iHMC using buffalo cytoplasts and differentiated somatic cells from cattle and goat and that the source of donor nucleus affects the developmental competence of interspecies embryos. [Copyright &y& Elsevier]

Abstract: Fertility in dairy cows has declined over the past five decades as milk production per cow has increased. Many hypotheses have been proposed to explain this including issues of genetics, physiology, nutrition and management, and these factors have been investigated at the animal, organ and cellular level at critical time points of the productive life of dairy cows. This paper reviews the physiological events and their causes and consequences affecting fertility in dairy cows and summarises these in a downloadable poster. We consider the following points to have the greatest negative impact on fertility and that they need to be prioritised in efforts to ameliorate the problem (others have been included in the review). Firstly, minimise negative energy balance and resolve any infection of the post partum uterus. Secondly, expression and detection of oestrus followed by insemination with high quality semen (day 0). Thirdly, ovulation and fertilisation of a high quality oocyte (day 1). Fourthly, an early increase in progesterone secretion from the corpus luteum (days 3–7). Fifthly, the uterine endometrium must produce an early and appropriate environment to stimulate embryo development (days 6–13). This leads to sixthly, a large embryo producing adequate quantities of interferon tau (days 14–18) that alters uterine prostaglandin secretion and signals maternal recognition of pregnancy (days 16–18). Future strategies to improve dairy cow fertility are needed for the benefit of the dairy industry and for cow welfare and should be based upon an integrative approach of these events. [Copyright &y& Elsevier]

Abstract: We previously showed that circadian genes clock, bmal1, cry1, cry2, per1, and per2 are expressed and function as maternal mRNA regulating events in the oocytes and preimplantation embryos of mice. Recent evidence indicates however that either or both expression profiles of circadian genes in some tissues, and transcript sequences of circadian genes, differ to generate the physiological differences between diurnal and nocturnal species. We therefore investigated the expression profiles of circadian genes in oocytes and preimplantation embryos of species other than mice, namely cattle and rabbits, representing diurnal and nocturnal species, respectively, and determined the protein sequences of circadian genes in these species. Quantitative real-time PCR revealed that all circadian genes considered in this study were present in the oocytes and preimplantation embryos of both species, and the transcript amounts of clock, cry1 and per1 contained in oocytes were significantly higher than in preimplantation embryos of both species. The transcripts of clock, cry1, and per1 of cattle and rabbits were determined by primer walking, and functional domains in the estimated amino acid sequences were compared between cattle and rabbits and with those of humans and mice. The sequences of clock, cry1, and per1 in cattle and rabbits closely resembled those in mice (85–100% homologies), and no difference based on diurnality or nocturnality was observed. These findings suggest that circadian genes in the oocytes and preimplantation embryos of mammals fulfill the same functions across species as maternal mRNA. [ABSTRACT FROM AUTHOR]

Abstract: In vitro embryo production (IVP) has been suggested to result in a greater proportion of male calves, longer gestation and heavier offspring than artificial insemination in Bos taurus cattle. Despite the increasing use of IVP in tropical countries, its effects upon these traits in Bos indicus have not been conclusively investigated. Gyr is a B. indicus dairy breed with known physiological differences from B. taurus, such as a longer gestation period and lighter offspring. The aim of this study was to evaluate the effect of IVP on gestation length, birth weight and gender ratio in Gyr offspring. Oocytes were recovered from Gyr cows by ovum pick-up and were matured and fertilized with thawed Gyr semen in vitro. Embryos were cultured in CR2aa medium with cumulus cells and 10% fetal calf serum under 5% CO2 at 38.5°C in air. Seven- to eight-day blastocysts were transferred to synchronized recipients. Data on gestation length and birth weight of calves from in vitro-produced embryos were compared to data obtained from Gyr calves produced by artificial insemination (AI) and natural breeding (NB) during the same period using analysis of variance, and the gender ratio was compared to the expected 1:1 ratio using a chi-square test. IVP increased (P <0.01) the percentage of male offspring (76.9%) compared to the expected 1:1 ratio, while no difference (P >0.05) was observed in the AI and NB groups. Gestation length was similar (P >0.05) between the IVP and AI groups, but IVP-derived offspring were heavier (P <0.05) than AI- and NB-derived ones, mainly for male calves (P <0.05). These data show that in vitro production affects the subsequent development of Gyr embryos, resulting in a skewed sex ratio and increased birth weight. [Copyright &y& Elsevier]

Abstract: The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5μL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3min and 30s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3min, and transferred into warming solution 2 for 1min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, P <0.05). In experiment 2, there was no significant effect of volume in hatching rates (58.3% 1μL, 61.3% 0.5μL and 80.5% control, P <0.05). In experiment 3, the composition of the holding medium of warming solution influenced hatching rates (84.1% TCM-199, 74.8% PBS and 91.1% control P <0.05). These data suggest that neither glass capillaries nor reduced sample volume could improve hatching rates after vitrification–warming with open pulled straw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected. [Copyright &y& Elsevier]

Abstract: Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150μg d-cloprostenol. On Day 7, all follicles >3mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14d. Each experiment had three treatment groups. In Experiment 1 (n =12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28d. Heifers from Group 1X-EB also received 2mg EB i.m. immediately after each OPU session. In Experiment 2 (n =11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos. [Copyright &y& Elsevier]

Abstract: The usefulness of IVF as a potential tool to evaluate the field fertility of bulls is equivocal and growth factor addition to culture media research is needed to delineate components needed for providing defined environments for embryos. The overall aim was to evaluate the in vitro development of embryos derived using a serum supplemented and serum-free production systems and semen from two bulls of different field fertility. The study was conducted to determine the combinatorial effect of stem cell factor (SCF) and/or insulin-like growth factor-I (IGF-I) in culture on subsequent embryo development in cattle. Oocytes were aspirated separately from ≥3 to <3mm follicles to test different follicle size populations and were matured in TCM-199 supplemented with LH, FSH, estradiol and BSA (Fraction V). Matured oocytes were fertilized in BSA supplemented synthetic oviductal fluid (SOF)-IVF medium. Presumptive zygotes were cultured for 8d (in humidified 5% CO2 at 38.5°C) in BSA supplemented SOF-in vitro culture (IVC) medium. SOF-IVC medium was supplemented with fetal bovine serum (4%), IGF-I (100ng/mL), SCF (50ng/mL) or IGF-I (100ng/mL)+SCF (50ng/mL). The development competence of embryos did not differ between the bulls and among the culture environments. Nevertheless, there was an effect of follicle size on cleavage rate (P <0.05) and a greater cleavage rate resulted from oocytes aspirated from ≥3mm follicles (71.0±1.5%) compared to those collected from <3mm follicles (64.8±1.6%). The overall cleavage rate (%); blastocyst formation (%); and expanded/hatched blastocyst formation (%) were 68.2±1.5 and 67.7±1.7; 29.4±1.4 and 28.6±1.5; and 18.6±1.2 and 18.5±1.1, respectively, for the bull of above and below average field fertility. The results indicate that follicle size for oocyte aspiration is effective for determining IVC success and that IVF may not discriminate among bulls of different field fertility. [Copyright &y& Elsevier]

Abstract: Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/H19 allelic expression. The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O’Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves. [Copyright &y& Elsevier]

Abstract: The reliable collection of peri-implantation embryos in the bovine has important ramifications to post-transfer consequences, particularly in the elucidation of mechanisms associated with post-hatching embryo development and to perturbations in developmental growth following transfer. This study analyzed both in vitro produced (IVP) and somatic cell nuclear transfer (SCNT) embryo-like structures (ELS) recovered at Day (D) 14 and D21. The recovered ELS were subsequently processed for histological examination. At D14 and D21, many of the embryos recovered in the IVP group conformed to the appropriate stage of development. However, a significant number of anomalies were present in the SCNT groups when examined in more detail. Histological examination revealed that irrespective of whether these embryos had undergone trophoblast expansion to an ovoid, tubular or filamentous morphology, many had a degenerated hypoblast layer and a large proportion did not possess an epiblast and therefore could not differentiate into any of the three germ layers as would be expected at the neural groove or somite stage. The prevalence of this developmental pattern was random and did not correlate with treatment (IVP or SCNT) or with types of structures recovered. The rapid embryo elongation period also coincides with the time of greatest embryonic loss and these observations could have important implications for assessing the recovery of embryos post-transfer where incorrect morphological assessment could lead to false implantation and pregnancy determination rates. The implementation of additional methodology is required to adequately characterize the quality of IVP and SCNT-derived embryos collected post-transfer. [Copyright &y& Elsevier]

Abstract: Bovine herpesvirus 1 (BoHV-1) is widely distributed among cattle populations and has been associated with cells, fluids, and tissues collected from donor animals for use in reproductive technologies. The purpose of this study was to determine if lactoferrin would inhibit BoHV-1 in cell culture and to evaluate if embryos could develop normally when cultured in vitro with lactoferrin. In Experiment 1, lactoferrin (10mg/mL) inhibited up to 25,000 plaque forming units (PFU)/mL of BoHV-1 in Madin Darby bovine kidney (MDBK) cell culture. In Experiment 2, lactoferrin (10mg/mL) combined with cidofovir (62.5μg/mL) inhibited up to 100,200PFU/mL of virus in cell culture. In Experiment 3, following fertilization, presumptive zygotes were cultured in media containing lactoferrin (10, 5, and 2.5mg/mL). Embryonic development and quality were assessed, and embryonic viability was determined by counting the nucleated cells of developed blastocysts. While lactoferrin did not affect the nucleated cell count of the treated embryos, it did significantly decrease blastocyst development. In conclusion, lactoferrin from bovine milk can inhibit BoHV-1 in cell culture. However, supplementation of in vitro culture medium with lactoferrin inhibits blastocyst development of in vitro-produced embryos. [Copyright &y& Elsevier]

A pathogen which has been shown to commonly contaminate in vitro bovine embryo production system is bovine pestivirus (bovine viral diarrhea virus). Three experiments were designed to evaluate the in vitro maturation (experiment I), fertilization (experiment II) and embryo development (experiment III) of immature oocytes, inseminated oocytes and presumptive zygotes in the presence of a bovine pestivirus (non-cytopathic, nCP type 1). The virus inoculum used was derived from a persistently infected cow. In experiment I, follicular oocytes (n=1257) recovered from slaughterhouse derived ovaries were randomly assigned to either a control group (n=578) which did not become exposed to bovine pestivirus and a treatment group (n=679) which was inoculated with bovine pestivirus (2.20–3.69 log10 TCID50/50 μl) at the time of commencement of in vitro maturation. Overall, there was no significant difference between the control and pestivirus inoculated oocytes in either the cumulus cell expansion rate (79±7.5% versus 74±10.7%) or the nuclear maturation rate (89±4.8% versus 85±7.4%), respectively. In experiment II, in vitro matured oocytes (n=607) were inseminated either in the absence (control; n=301) or the presence of bovine pestivirus (4–4.6 log10 TCID50/50 μl; n=306). A significant (P<0.01) reduction in the overall number of fertilized oocytes with two well formed male and female pronuclei was observed in the treatment group compared to the control group (58.5±5.8% versus 73.3±3.6%, respectively). In experiment III, after in vitro maturation and fertilization, presumptive zygotes were randomly assigned to either a control group (n=139) which was not exposed to bovine pestivirus or a treatment group which was inoculated with bovine pestivirus (2.97–4.47 log10 TCID50/30 μl; n=139). The zygotes were then cultured under mineral oil in an atmosphere of 88% N2, 7% O2 and 5% CO2 at 39 °C. The morphologic appearance of the embryos was assessed 48 h after the commencement of culture, and then every 48 h up to days 7–8 after insemination. The 22% (31/139) and 3.6% (5/139) of the presumptive zygotes developed to the morula or blastocyst stage in the control and the bovine pestivirus inoculated groups, respectively (P<0.001). This study demonstrates that bovine pestivirus has a significant detrimental effect on in vitro fertilization and early in vitro embryo development. [Copyright &y& Elsevier]

The objective of this study was to evaluate the effects of oocyte quality on in vitro development and the level of transcriptional activity in early bovine embryos. Cumulus-oocyte complexes (COC) were divided into six classes based on their cumulus investment and on the texture of the ooplasm. Embryos originating from oocytes with more than five layers of cumulus cells and with slight expansion of the cumulus and/or granulation in the ooplasm (class II) developed to the blastocyst stage as frequently as embryos originating from oocytes of class I which showed no signs of atresia (13.9 and 13.7% for classes I and II, respectively). Oocytes with fewer than five layers of cumulus cells and homogeneous ooplasm (class III) had lower cleavage (63.1%) than oocytes with more than five layers of cumulus cells (77.2 and 83.6% for classes I and II, respectively); however, development to the blastocyst stage was similar (12.7%). More advanced atresia such as the presence of granulations in oocytes with less than five layers of cumulus cells (class IV), the absence of cumulus (class V), or the presence of expanded cumulus with dark clumps (class VI) reduced cleavage (57.4, 35.9 and 56.3% for classes IV–VI, respectively) and blastocyst formation (3.5, 0.5 and 1.9% for classes IV–VI, respectively). We examined the effects of oocyte quality on the level of transcriptional activity in in vitro produced embryos at the 2-, 4-, 8-, 16-cell stage and in embryos remaining at the 8-cell stage while the majority had progressed to the 16-cell stage (8-cell delayed embryos) by labeling with 3H-uridine followed by RNA precipitation and scintillation counting. For each developmental stage, there was no significant effect of oocyte class on uptake and incorporation of 3H-uridine into embryos, with the exception of uptake at the 8-cell stage which was higher (P<0.05) in embryos from class V–VI oocytes. Labeled uridine uptake (in embryos from classes I–II and III oocytes) and incorporation (in embryos from oocytes of all classes) increased significantly at the 16-cell stage compared to earlier stages. Eight-cell delayed embryos originating from classes I to IV oocytes incorporated significantly more 3H-uridine than normally developing 8-cell embryos. In conclusion, these results expand on previous work showing that oocytes with early signs of atresia have good development potential. No differences in transcriptional activity in embryos originating from different classes were detected. However, the results obtained with 8-cell delayed embryos indicated that transcriptional activity was determined by the interval after fertilization rather than the number of cell cycles. [Copyright &y& Elsevier]