[Show abstract][Hide abstract] ABSTRACT: Whole plant corn silage is an important component of dairy cow nutrition. Growing conditions in a moderate climate, as are present in large parts of Europe and North America, are often beneficial for mould contaminations to occur. Very commonly, varieties of Fusarium spp. grow on the corn plant producing the non-steroidal estrogenic mycotoxin zearalenone (ZEN). Numerous studies have shown that ZEN massively interacts with the porcine reproductive system. Nevertheless, only a few studies have assessed the possible effects on dairy cows, most publications being field studies following accidental exposure. Therefore, it was aim of the present study to assess the effects of a controlled dietary exposition on selected reproductive parameters in dairy cows. The chosen exposure concentration was within the range of the 'Commision Recommendation on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and fumonisins in products intended for animal feeding' of the European Union. Corn plants were artificially inoculated with Fusarium spp. and the silage obtained from the whole plant was analysed to determine to which degree the silage contained ZEN. Thirty Holstein Friesian cows and heifers (aged between 2 years 3 months and 5 years at calving) were allocated to 3 groups with 10 animals each: control (CTL), uncontaminated silage; ZEN 50, silage with 0.25mg of ZEN/day; and ZEN 100, silage with 0.5mg of ZEN/day. The contaminated rations were distributed by automated feeding stations to ensure an equal uptake. Feeding began 7 days post-parturition. Blood samples were obtained twice weekly for 12 weeks and were analyzed regarding the progesterone level via radioimmunoassay. At the same time, the oestrous cycle was monitored by ultrasonography. Furthermore, the size of the corpus luteum (CL) was measured 7±2 days after the third ovulation. Simultaneously, the luteal blood flow was documented via Doppler ultrasonography. The peripheral blood progesterone level (xgeom.±CV; CTL: 9.3±1.7; ZEN 50: 8.5±1.6; ZEN100: 7.1±1.5) did not differ among groups. Furthermore, it was not possible to detect differences in luteal blood flow, size of the CL (mean±SD; CTL: 6.4±1.7cm(2); ZEN50: 5.9±1.3cm(2); ZEN 100: 5.0±1.0cm(2)) or the ratio of luteal size and size of the area supplied with blood (CTL: 0.1±0.1; ZEN 50: 0.1±0.0; ZEN 100: 0.1±0.1). Although the mean length of the oestrous cycles (n=60; distributed among groups) did not differ among groups (CTL: 24.0±1.7 days; ZEN 50: 27.2±1.6 days; ZEN 100: 27.7±1.4 days), it was possible to detect an interaction between the age of the animal and the treatment regarding the cycle length in tendency (P<0.07) by two-way ANOVA. The results of the present study indicate that, independent of the age of the animal, exposure to ZEN even within recommended dosages might result in an endocrine disruption in dairy cows. A larger sample size would contribute to the verification of these findings. Further analyses regarding the peripheral level of 17β-estradiol as well as molecular examinations are under progress.
Reproduction Fertility and Development 12/2013; 26(1):149-50. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor 1 (IGF1) is essential for regulating physiological processes such as growth and development of fetal and placental tissues (Bauer et al. 1998, Fowden 2003). During early embryonic development, IGF1 plays an important role, as it leads to a reduction of apoptosis and decreases early embryonic mortality (Block et al. 2007). The signal transduction of IGF1 is carried out by its specific binding to the membrane-embedded insulin-like growth factor 1 receptor (IGF1R). The expression of IGF1R is a potential quality marker of in vitro produced embryos (Liu et al. 1997, Yaseen et al. 2001). Thus far, analysis of the relative amount of specific transcripts is the method of choice to study bovine pre-implantation embryos, as information on protein expression is scarce. Therefore, it is of great interest to analyse protein expression and to determine if and to which extent these results differ from results obtained in previous mRNA expression analyses. In the present study, a total of 4800 cumulus-oocyte-complexes were deployed in 60 in vitro produced runs. The cleavage rates averaged 57.4±7.3% and blastocyst rates were 27.6±7.5% at Day 8 of culture. Embryos at the blastocyst stage were frozen and stored at -80°C for further experiments. The protein expression of the IGF1R during early embryonic development was investigated by Western blot analysis testing 8 different antibodies. Seven of these antibodies were commercially available and mainly not tested in the bovine species. Only 1 of these antibodies resulted in a weak signal for the IGF1R protein in bovine blastocysts. Therefore, a specific peptide antibody against 2 peptide sequences of the α unit of the bovine IGF1R was produced. The analysis of the IGF1R protein with this antibody resulted in the determination of a signal in a pool of 100 blastocysts, which was weaker than in the positive control (20μg of bovine liver protein extract). The detection of the IGF1R protein localization was possible in all different stages of embryonic development from the zygote to the expanded blastocyst using immunfluorescence staining with the specific peptide antibody. The IGF1R protein was mainly expressed in the plasma membrane of single blastomeres and also weakly in the cytoplasm. As the early bovine embryo expresses IGF1R throughout all stages, the main function of IGF1 in embryonic development needs to be further elucidated.
Reproduction Fertility and Development 12/2013; 26(1):149. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Progesterone (P4) is important for the developmental competence of cumulus-oocyte complexes (COC) used for in vitro maturation (IVM). In a recent study, we were able to show that circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated ovum pickup sessions, which might affect further development. (Schlüter et al. 2013 Reprod. Fertil. Dev. 25, 250). The aim of the present study was to determine the influence of different P4 concentrations during IVM on the molecular quality of bovine COC. The COC were collected from slaughterhouse ovaries and cultured as described recently (Stinshoff et al. 2011 Theriogenology 76, 1433-1441). The IVM medium was supplemented with 0, 50, 150, 300, and 450ngmL(-1) P4. Ethanol served as vehicle control. After IVF with a bull of proven fertility, the presumptive zygotes were cultured in SOF under 5% oxygen (Stinshoff et al. 2011). Cleavage and developmental rates were determined at Day 3 and Day 7/8 (Day 0: IVF). Additionally, maturation rates were assessed. For mRNA analysis, immature and matured denuded COC (n=5) were individually frozen at -80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), nuclear progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using ANOVA followed by multiple pairwise comparisons using Tukey's test. A P-value of <0.05 was considered significant. The percentage of oocytes that reached the MII stage was similar in oocytes from all treatment groups (82.2-88.1%). Despite similar cleavage rates across all groups, the developmental rates did show significant differences. More embryos developed to the blastocyst stage stemming from oocytes cultured without any supplement or cultured only with alcohol compared to oocytes stemming from the group cultured with less than 50ngmL(-1) P4 (25.4±5.7 and 27.9±7.2 v. 15.8±2.6). The relative abundance of SCL2A1, BMP15, and PGRMC1transcripts in single oocytes did not show differences related to the supplementation of the IVM medium, whereas GDF9, HIF2α, and PAQR5 mRNA was reduced in oocytes of all groups compared with immature ones. The PGR and PGRMC2 transcripts were increased in matured oocytes of the control group and the vehicle control group (PGRMC2). In summary, supplementation of the IVM medium with different P4 concentrations had an effect on the molecular quality of oocytes after IVM, which might affect further development.
Reproduction Fertility and Development 12/2013; 26(1):171. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vitro production (IVP) of bovine embryos has been improved immensely throughout the last decades. Nevertheless, embryos generated in vitro still differ from their in vivo-produced counterparts. It is possible to achieve blastocyst rates of up to 70% if in vivo-matured oocytes are used. In contrast, if oocytes are matured in vitro, blastocyst rates are only half that of those matured in vivo. This rather limited success may be attributed to the heterogeneous population of oocytes which are normally retrieved from follicles of 3-8 mm rather than from preovulatory follicles. In contrast to the in vivo-ovulated oocyte, these oocytes lack development up to the preovulatory stage and are matured in vitro. Therefore, much effort has been devoted to the establishment of non-invasive and non-perturbing means for selecting the most competent oocytes, for example the extensiveness and compactness of the cumulus-corona investment and the granulation of the ooplasm. In vitro culture (IVC) conditions have been enhanced in the last few years, mainly by adjustment of media formulations, whereas the in vitro maturation (IVM) protocols stay invariable. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting matured oocyte. The scope of this review is to give an overview of the current situation of in vitro maturation of mammalian oocytes with emphasis on the bovine species. Special attention has been paid to the in vivo situation in the follicle and how a better understanding of these intrafollicular factors will aid to improve the in vitro maturation conditions.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to investigate the effect of time of first postpartum ovulation after calving on uterine involution in dairy cows with and without uterine puerperal disease. Transvaginal follicular puncture (FP) of follicles >6 mm suppressed ovulation and development of a CL until Day 42 after calving. Fifty-three lactating Holstein Friesian cows (3.4 ± 1.2 years old, parity 2.5 ± 1.0 [median ± mean absolute deviation]) were divided into groups on the basis of the presence (UD+) or absence (UD-) of uterine disease and whether FP was carried out (FP+) or not (FP-). Uterine disease was defined as the occurrence of retained fetal membranes and/or metritis. This resulted in the following groups: UD-FP- (n = 15), UD-FP+ (n = 13), UD+FP- (n = 13), and UD+FP+ (n = 12). A general examination, vaginoscopy, transrectal palpation, and transrectal B-mode sonography of the reproductive organs were conducted on Days 8, 11, 18, and 25 and then every 10 days until Day 65 after calving. After hormonal synchronization of ovulation (cloprostenol between Days 55 and 60 postpartum and GnRH 2 days later), cows were inseminated in the next spontaneous estrus. On average, the cows ovulated on Day 21.0 ± 6.0 (UD-FP-), 50.0 ± 4.0 (UD-FP+), 16.0 ± 3.0 (UD+FP-), and 48.0 ± 2.0 (UD+FP+) postpartum. Calving-to-conception interval and first-service conception rates were not affected by FP (P > 0.05). Healthy cows with FP had smaller (P < 0.05) uterine horn and cervical diameters assessed sonographically than cows without FP. FP reduced the prevalence of purulent vaginal discharge and uterine size assessed transrectally in UD+ cows (P < 0.05). The results showed that suppression of an early ovulation by transvaginal FP improved uterine involution in cows with and without uterine disease.
[Show abstract][Hide abstract] ABSTRACT: The somatotropic axis is a key metabolic pathway during transition from late pregnancy to early lactation in dairy cows. The first objective of this study was to determine the feasibility of selecting cows with persistent differences in total insulin-like growth factor 1 (IGF-1) concentration by taking only a single antepartum blood sample. The second objective was to elucidate the underlying causes of differences in peripheral IGF-1 concentrations throughout late pregnancy and whether hormonal axes also differed in dairy cows with low versus high IGF-1. Twenty clinically healthy Holstein Friesian cows were chosen based on their plasma IGF-1 concentration at 244 to 254 d after artificial insemination (AI) and other selection criteria (health status, body condition score, number of lactations). These cows were selected from a large-scale farm, transported to the clinic, and monitored daily from 261 to 275 d after AI. The concentrations of IGF-1, growth hormone, IGF binding proteins 2, 3, and 4, insulin, cortisol, thyroid hormones, progesterone, and estradiol were measured. Ultimately, 7 IGF-1-low and 7 IGF-1-high cows were statistically analyzed. Additionally, a liver biopsy was taken on d 270 ± 1 after AI for analysis of gene expression of somatotropic family members, liver deiodinase 1, and suppressor of cytokine signaling-2. It was possible to select cows with different IGF-1 concentrations based upon only 1 blood sample collected in late pregnancy. Concentrations of IGF-1 in IGF-1-low versus IGF-1-high animals (n = 7 each) remained significantly different between groups from the day of selection of the animals until d 275 after AI. Second, the differences in total plasma IGF-1 concentration between experimental groups may be attributed to differences in hepatic production of acid labile subunit. The ability of IGFBP-3 to bind IGF-1 declined before calving in all cows. Furthermore, in addition to decreased mRNA expression of growth hormone receptor 1A and IGF-1 relative to calving, serum binding capacities for IGF-1 also decreased. Insulin-like growth factor binding protein 4 mRNA expression was higher in cows with low IGF-1 concentrations; this binding protein inhibits IGF-1 action at the tissue level and therefore may reduce IGF-1 bioavailability. Finally, other endocrine end points (e.g., insulin and thyroid hormones) differed between the 2 groups.
Journal of Dairy Science 04/2013; · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is well documented that global warming is unequivocal. Dairy production systems are considered as important sources of greenhouse gas emissions; however, little is known about the sensitivity and vulnerability of these production systems themselves to climate warming. This review brings different aspects of dairy cow production in Central Europe into focus, with a holistic approach to emphasize potential future consequences and challenges arising from climate change. With the current understanding of the effects of climate change, it is expected that yield of forage per hectare will be influenced positively, whereas quality will mainly depend on water availability and soil characteristics. Thus, the botanical composition of future grassland should include species that are able to withstand the changing conditions (e.g. lucerne and bird's foot trefoil). Changes in nutrient concentration of forage plants, elevated heat loads and altered feeding patterns of animals may influence rumen physiology. Several promising nutritional strategies are available to lower potential negative impacts of climate change on dairy cow nutrition and performance. Adjustment of feeding and drinking regimes, diet composition and additive supplementation can contribute to the maintenance of adequate dairy cow nutrition and performance. Provision of adequate shade and cooling will reduce the direct effects of heat stress. As estimated genetic parameters are promising, heat stress tolerance as a functional trait may be included into breeding programmes. Indirect effects of global warming on the health and welfare of animals seem to be more complicated and thus are less predictable. As the epidemiology of certain gastrointestinal nematodes and liver fluke is favourably influenced by increased temperature and humidity, relations between climate change and disease dynamics should be followed closely. Under current conditions, climate change associated economic impacts are estimated to be neutral if some form of adaptation is integrated. Therefore, it is essential to establish and adopt mitigation strategies covering available tools from management, nutrition, health and plant and animal breeding to cope with the future consequences of climate change on dairy farming.
[Show abstract][Hide abstract] ABSTRACT: It is known that the progesterone (P4) provided by the corpus luteum is essential for the maintenance of pregnancy. It has been suggested that supplying external P4 in vivo is beneficial to the establishment and upkeep of pregnancy. The aim of the present study was to assess the effects of supplementation with different concentrations of P4 on either of 2 days of in vitro culture (IVC) on early bovine embryo development in an in vitro model. A total of 5073 cumulus-oocyte complexes were matured and fertilized in vitro. Before culture, they were collected in groups of 30 and allocated to 1 of 9 groups. The groups were supplemented with 10, 20, or 100ng of P4 on Days 4 or 5 of IVC (IVF=Day 0). Alcohol (ETOH) was used as the solvent, so 8µL of ETOH was used per supplementation. Therefore, two additional groups were supplemented with only ETOH on Day 4 or 5 of IVC. The presumptive zygotes allocated to group 9 were not supplemented. A culture system without oil overlay was used to prevent the lipophilic P4 from moving into the oil. Embryo cleavage and development rates were determined solely on Day 8 of IVC. Single expanded blastocysts were stored at -80°C for RT-qPCR. Subsequently, the relative amounts of six developmentally important gene transcripts (IGF1R, SLC2A1, HSD3B1, IFNT, PGRMC1, and PGRMC2) were analysed in single embryos of all groups. Statistical analysis was performed using one-way and two-way ANOVA, and the level of significance was set at P≤0.05. Cleavage and development rates did not differ among groups (see Table 1). The relative abundance of IGF1R, SLC2A1, PGRMC1, and PGRMC2 was not affected by either the concentration or the timing of P4 supplementation. Nevertheless, there was a statistically significant interaction between the day of treatment and the concentration used for the expression of HSD3B1 mRNA. When 20ng of P4 was added on Day 5 of IVC, significantly more HSD3B1 transcripts were detected than if 10ng, 100ng, or ETOH alone was added. The expression of IFNT was not affected by the day of supplementation, only by the concentration used. Thus, supplementation with 20ng of P4 resulted in a significantly higher level of transcripts than when 10ng or ETOH was supplemented. The results indicate that the amount of P4 present during early embryonic development and the timing of its presence had an impact on molecular developmental competence. However, no effects concerning morphological development up to the blastocyst stage could be detected.
Reproduction Fertility and Development 12/2012; 25(1):265. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The developmental competence of cumulus-oocyte complexes (COC) used for in vitro production is dependent on several factors including the stage of the oestrus cycle. In a recent study, we were able to show that circulating progesterone (P4) had no effect on follicle number, size, recovery rate, or in vitro production suitability of recovered COC (Schlüter et al. 2012 Reprod. Fertil. Dev. 24, 175-176). The aim of the present study was to determine the influence of circulating P4 concentrations on the molecular quality of bovine COC collected during repeated OPU sessions. The COC were aspirated twice per week for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle after spontaneous ovulation (ovulation=Day 0). Blood samples were taken at the time of each OPU session, and P4 concentrations were determined using a radioimmunoassay. All animals showed clinical signs of oestrus and large follicles (≥8.5mm) during the course of the OPU sessions. Following the aspiration of a large follicle, a CL-like structure (induced CL) could be detected. According to the P4 concentrations, the cycle was divided into 3 phases: CL phase after spontaneous ovulation (oCL; P4: ≥1ngmL(-1)), follicle phase 1 (Fp; P4 <1ngmL(-1)), and induced CL phase (iCL; P4: ≥1ngmL(-1)). The length of the cycle after spontaneous ovulation did not differ significantly from that after induced ovulation (22.4±3.1 days v. 23.8±1.8 days, respectively). During the oCL-phase, blood P4 concentrations were significantly higher than during the iCL-phase (4.9±2.3ngmL(-1) v. 3.0±1.6ngmL(-1)). For mRNA analysis, denuded COC were individually frozen at -80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey's test. A P-value of ≤0.05 was considered significant. The relative abundance of all transcripts except SCL2A1 was significantly increased in oocytes collected from follicles of the oCL phase compared with that from oocytes that had been aspirated during the iCL phase. A significant increase in the relative amount of PGR, PGRMC1, PGRMC2, and BMP15 transcripts was detected in oocytes stemming from the follicular phase to those from the iCL phase. No differences in the relative abundance of all transcripts were seen comparing oocytes from oCL phase and oocytes from the follicular phase. In summary, circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated OPU session, which might affect further development.
Reproduction Fertility and Development 12/2012; 25(1):250. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Summary The objectives of this research were to study the influence of a reduced oxygen concentration during in vitro maturation (IVM) and examine the effect of follicular glucose concentration on bovine in vitro development and sex distribution. In the first experiment, abattoir-derived cumulus-oocyte complexes (COC) were matured under 5% O2 or 20% O2. Secondly, COC were isolated and the glucose (G) concentration of each follicle was determined. COC were pooled in groups (G<1.1mMol or G≥1.1mMol) according to the glucose content before being subjected to in vitro production (IVP). Cleavage and development rates were assessed on days 3, 7 and 8 post insemination. Blastocysts of each group were sexed by polymerase chain reaction (PCR). Expanded blastocysts were stained to assess total cell numbers and live-dead cell ratio. Cleavage and development rates stayed similar after reducing the O2 concentration during IVM. The sex ratio of embryos generated from oocytes matured under 5% O2 was shifted in favour of the female (♀: 61.9%), whereas the sex ratio of embryos belonging to the IVM 20% O2 group did not differ significantly from the expected 50:50 ratio. Neither a 'higher' nor a 'lower' intrafollicular glucose concentration influenced cleavage and development rates, cell numbers or live-dead cell ratio. Eighty five per cent (G<1.1) and 63.6% (G≥1.1) of the analysed embryos were female. In summary, neither a reduced O2 concentration during IVM nor selection based on follicular glucose concentrations affected the morphological quality of embryos. Although the sex distribution was shifted in favour of female embryos in all three experimental groups, more male embryos could be seen in the G≥1.1 group compared with the G<1.1 group.
[Show abstract][Hide abstract] ABSTRACT: Although sex-sorted sperm have been used for AI and IVF for over a decade there is still need to improve the technology as the results are highly variable. The goal of the present study was to assess the effect of seminal plasma and seminal plasma proteins as a supplement to sorted sperm on subsequent embryonic development, as a beneficial effect of these substances has been reported. In vitro matured oocytes were fertilized in vitro with either unsorted sperm (n = 215; Group 1), bulk sorted sperm (n = 226; Group 2), bulk sorted sperm extended in the presence of 1% seminal plasma (n = 185; Group 3) or bulk sorted sperm supplemented with seminal plasma proteins (4 mg mL(-1); n = 254; Group 4). An additional group of oocytes (n = 307; Group 5) was fertilized with the semen of another bull routinely used for IVF and served as a laboratory standard control. Subsequently, the presumptive zygotes were cultured for 8 days under standard conditions (SOFaa, 39 °C, 5% CO(2), 5% N(2)). Cleavage rates were assessed on day 3 p.i. (post insemination; group 1: 30.5 ± 14.7%; group 2: 28.8 ± 9.8%; group 3: 20.8 ± 14.9%; group 4: 25.7 ± 8.2%; group 5: 54.8 ± 11.5%). Development rates were documented on days 7 p.i. (group 1: 7.3 ± 6.6%; group 2: 5.6 ± 3.1%, group 3: 6.2 ± 7.7%, group 4: 6.7 ± 5.9%, group 5: 20.2 ± 6.9%) and 8 p.i. (group 1: 8.9 ± 7.0%; group 2: 6.0 ± 2.9%; group 3: 8.6 ± 11.3%; group 4: 7.8 ± 6.2%; group 5: 23.3 ± 7.8%), respectively. Significant differences among cleavage and development rates could only be seen for Group 5 compared to all other groups. However, this difference between Groups 1-4 vs. Group 5 regarding the development rates on Day 8 could not be detected when assessing the development rates on base of the number of cleaved embryos instead of the number of oocytes fertilized (group 1: 31.4 ± 17.2%; group 2: 26.0 ± 21.0%; group 3: 33.3 ± 19.05%; group 4: 26.6 ± 17.8%; group 5: 42.6 ± 11.3%). The relative abundance of six different developmentally important gene transcripts (G6PD, HSP1A1, SLC2A3, BAX, BCL2L1, DNMT3A) was determined using single Day 8 expanded blastocysts of all five groups. No significant differences were seen among the embryos of the five groups. Our results show that neither the bulk sorting procedure nor the addition of seminal plasma or seminal plasma proteins, respectively, affected cleavage and development rates when sperm from a specific bull was used. Additionally, sorting and subsequent exposure of sperm to either seminal plasma or seminal plasma proteins did not influence mRNA expression in bovine IVP embryos.
[Show abstract][Hide abstract] ABSTRACT: Shortly after parturition the metabolic situation of high-yielding dairy cows is often dominated by a negative energy balance. These effects affect the whole animal and may especially be detected in the reproductive tract, where they result in reduced fertility. An oral supplementation with dietary fats is often used to counteract by reducing milk fat content and, thus, supplying the individual animal with an increased amount of energy. The focus of the present study was to analyse the effects of an oral supplementation with conjugated linoleic acids (CLA) on corpus luteum (CL) function. Healthy Holstein-Friesian cows and heifers were randomly allocated to 2 treatment groups (Group 1: 50g of CLAday(-1) per animal, 2 heifers, 6 cows; Group 2: 100g of CLAday(-1) per animal, 2 heifers, 6 cows) and 1 control group (Ctl; 0g of CLAday(-1) per animal, 3 heifers, 4 cows). Feeding of the supplement began shortly after calving. After calving, all animals were subjected to a standard synchronisation protocol and experienced AI on Day 59±3. Following AI, transvaginal biopsies of the corpus luteum were obtained of pregnant (Group I: n=4; Group II: n=4; Ctl: n=4) and nonpregnant (Group I: n=4; Group II: n=4; Ctl: n=3) animals on Days 6, 13 and 20 post-AI. Animals deemed pregnant on Day 28 were again biopsied on Day 42. Additionally, blood samples were taken from the vena sacralis mediana at the time of each biopsy. The biopsies were analysed regarding the relative abundance of 8 gene transcripts (VEGF, ECE1, PLA2G4A, PTGS2, PTGFR, PPARG, STAR and HSD3B1) via RT-qPCR. Blood samples were analysed for their concentration of progesterone through a radioimmunoassay (RIA). Statistical analysis for both datasets was performed via a 3-way ANOVA with adjoining Tukey test. The expression of 7 of these genes was affected by 1, 2, or all 3 of the following factors: day of cycle (VEGF, ECE1, PLA2G4A, PTGFR, STAR and HSD3B1), pregnancy status (ECE1, PTGFR and HSD3B1) and CLA supplementation (ECE1, PTGS2, PTGFR, STAR and HSD3B1). The effects of the CLA supplementation could be seen as a down-regulation in the mentioned gene transcripts. Progesterone concentrations differed significantly in dependency of the pregnancy status (significantly higher in pregnant vs nonpregnant individuals) of the animals, as well as during the days of the oestrous cycle (physiological progesterone curve with highest values on Day 13 of these samples). An effect of the oral supplementation with CLA could be detected during the early luteal phase (Day 6) where animals that had received 100g of CLAday(-1) had a significantly lower blood progesterone concentration than those receiving 50g of CLAday(-1) or no CLA. In conclusion, dietary CLA supplementation has an effect on luteal gene expression and functionality.
Reproduction Fertility and Development 12/2011; 24(1):140-1. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Today, ovum pickup (OPU) followed by in vitro production (IVP) of bovine embryos is an integral part of many breeding programs. The quality of the obtained cumulus-oocyte complexes (COC) limits the success of embryo production. The developmental competence of the COC is dependent on several factors, including the stage of the oestrous cycle, the stage of follicular development and the follicular diameter. The aim of the present study was to determine the influence of circulating progesterone (P4) concentrations during repeated OPU sessions on the morphological quality of bovine COC. Cumulus-oocyte complexes were aspirated twice weekly for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle (ovulation=Day 0). During each session, number and diameter of the punctuated follicles and diameter, consistency and cavities in the corpus luteum (CL) were recorded. Follicles were assigned to 3 groups according to their diameter (3 to 5mm=small follicles; 6 to 8mm=intermediate follicles; >8mm=large follicles). Additionally, blood samples were taken at the time of each OPU session and blood P4 concentration was determined using a radioimmunoassay. The COC were categorised as IVP-suitable (round, ≥3 layers of cumulus cells, homo- or heterogeneous ooplasm) or unsuitable according to their morphological quality. All animals showed signs of oestrus accompanied by the presence of large follicles during the course of the OPU sessions. Statistical analysis was performed by an ANOVA followed by a Tukey test. A P-value of <0.05 was considered significant. The mean (±s.e.m.) cycle lengths for all heifers were 23.8±4.6 days. Following the aspiration of a large follicle, a CL-like structure could be detected (referred to as "induced CL"). According to the P4 concentrations, the cycle was divided into 4 phases: natural CL phase (nCL; P4 ≥1ngmL(-1)), follicle phase 1 (Fp1; P4 <1ngmL(-1)), induced CL phase (iCL; P4 ≥1ngmL(-1)), or follicle phase 2 (Fp2; P4 <1ngmL(-1)). During the nCL phase, blood P4 concentrations were significantly higher than during the iCL phase (4.9±2.3ngmL(-1), n=12 vs 3.0±1.6ngmL(-1), n=10). There were no differences in follicle numbers, the diameter distribution of follicles, recovery rates, or number of retrieved IVP-suitable COC (nCL: 3.1±3.4; Fp1: 3.3±3.7; iCL: 2.7±3.0; Fp2: 3.7±3.7; Table 1). In summary, circulating P4 concentrations had no effect on follicle number, diameter, recovery rate, or IVP suitability of recovered COC.
Reproduction Fertility and Development 12/2011; 24(1):175-6. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are still immense differences in the quality of in vitro produced embryos compared to their in vivo generated counterparts. These differences include a higher sensitivity of in vitro produced embryos towards cryopreservation. The quality of such embryos has been evaluated by morphological examination as well as the assessment of total cell numbers, pregnancy rates and in few cases through the analysis of their gene expression. The aim of the present study was to determine whether different cryopreservation methods have an influence on the quality of in vitro produced embryos after thawing. Bovine blastocysts were produced in a standard culture system (SOFaa). Having reached the stage of an expanding blastocyst on day 7, embryos were randomly either vitrified (n = 106) or cryopreserved conventionally (n = 131). Reexpansion (24h post thawing; 86/106 [81.1 ± 20.3%] of the vitrified embryos, 104/131 [79.4 ± 16.5%] of the conventionally cryopreserved embryos respectively) and hatching rates (48 h post thawing; 67/106 [63.2 ± 24.5%] of the vitrified embryos, 80/131 [61.1 ± 29.3%] of the conventionally cryopreserved embryos respectively) were similar. No significant differences in total cell numbers as well as live-dead cell ratio could be seen in hatched blastocysts of either cryopreservation group as well as in a control group (embryos cultured up to the stage of a hatched blastocyst and not cryopreserved by either method). Additionally, RT-qPCR was used to assess the relative abundance of eight developmentally important genes (HSPA1A, SLC2A1, SLC2A3, DSC2, CDH1, TJP1, DNMT3A, IFNT2) in hatched embryos of all groups. The results revealed significant differences in 4 of 8 (HSPA1A, SLC2A1, TJP1, DSC2) gene transcripts when comparing vitrified embryos to embryos of the control group and in 6 of 8 gene transcripts (HSPA1A, SLC2A1, TJP1, SLC2A3, DNMT3A, IFNT2) when comparing slow frozen embryos to embryos of the control group. Furthermore, the comparison of vitrified embryos to those frozen conventionally solely showed significant differences in the relative abundance of IFNT2. These results indicate that although not visible in gross morphology, but in the relative amount of gene transcripts, vitrification may be the more suitable method to cryopreserve in vitro produced bovine embryos cultured in SOF media.
[Show abstract][Hide abstract] ABSTRACT: Artificial insemination (AI) of sex-sorted sperm results in decreased fertility, compared with non-sorted sperm, in most species. However, this has not been the case in sheep, where the low-dose AI of sex-sorted ram sperm produced similar, if not superior, fertility to non-sorted controls. The aim of the present study was to determine the impact of sex-sorting technology on ovine embryo gene expression following embryo production in vivo and in vitro. After semen collection, ejaculates were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen. Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched sperm, or non-sorted control sperm, and collected by uterine flushing on Day 6 after AI. Embryos were produced in vitro using the same sperm treatments and cultured in vitro for 6 d. The relative abundance of selected gene transcripts was measured in high-grade blastocysts, defined by morphological assessment, using RT-qPCR. The mRNA expression of DNMT3A and SUV39H1 was upregulated in embryos cultured in vitro, compared to those cultured in vivo (DNMT3A: 3.61 ± 1.08 vs 1.99 ± 0.15; SUV39H1: 1.88 ± 0.11 vs 0.88 ± 0.07; mean ± SEM; P < 0.05). Both G6PD and SLC2A3 transcripts were reduced in embryos produced from sex-sorted sperm, in vivo (SLC2A3: 0.23 ± 0.03 vs 0.64 ± 0.10; G6PD: 0.32 ± 0.04 vs 1.01 ± 0.16; P < 0.05). The expression of DNMT3A was up-regulated in male (3.85 ± 0.31), compared to female embryos (2.34 ± 0.15; P < 0.05). This study contributes to the growing body of evidence citing aberrant patterns of gene expression resulting from in vitro culture. Whereas the process of sex-sorting altered the expression of several of the genes examined, no effect on embryo development was detected.
[Show abstract][Hide abstract] ABSTRACT: Production methods and culture systems have been shown to affect blastocyst mRNA expression and cryopreservability, which may serve as sensitive indicators of embryo quality and developmental competence. In the present study, the impact of four established culture conditions for producing bovine blastocysts (in vitro production, IVP; gamete intra-fallopian transfer, GIFT; transfer of cleaved stages into the oviduct, CLVT; multiple ovulation embryo transfer, MOET) was assessed, in terms of both cryosurvival and levels of mRNA expression of several selected genes (occludin, desmocollin 2, solute carrier family 2 member 3, BAX, BCL-XL, heat shock protein 1A, aquaporin 3, DNA methyltransferase 1a) detected with RT-qPCR. At 24 hours post-thawing, blastocysts derived from in vitro production showed a significantly higher re-expansion rate compared to the other groups. At later times, this difference was no longer significant. Before freezing, embryos of the MOET group showed significantly more desmocollin 2 mRNA compared to embryos produced using other culture methods. After freezing, significant upregulation was found in transcripts of heat shock protein 1A in embryos of all groups; of solute carrier family 2 member 3, only in IVP derived embryos; of BAX, BCL-XL, occludin, desmocollin 2, only in the MOET and IVP groups. Aquaporin 3 and DNA methyltransferase 1a were neither up- nor downregulated in blastocysts of any group. In conclusion, these findings suggest that, after freezing, embryos seem to have switched on mRNA synthesis, an active metabolism, operational cell connections, and are prepared for hatching and beyond.
[Show abstract][Hide abstract] ABSTRACT: Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.
Cytogenetic and Genome Research 01/2011; 132(4):239-47. · 1.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BlastocystmRNA expression and cryopreservability are thought to be suitable indicators of embryo quality and developmental competence and have been shown to be affected by production methods and culture systems. The aim of the present study was to assess cryosurvival and levels ofmRNA expression of selected genes [occludin, desmocollin 2, solute carrier family 2 member 3 (formerly glucose transporter 3), BAX, BCL xL, heat shock protein A1A (formerly heat shock protein 70.1), aquaporin 3, and DNA methyltransferase 1a] of bovine blastocysts derived by 4 different, established culture methods [in vitro production (IVP); multiple-ovulation embryo transfer (MOET); transfer into the heifer oviducts of gametes (GIFT); or in vitro derived cleaved stage embryos (Days 2-7)]. Linear models were used for the comparison of the relative abundances of the blastocystmRNA transcripts. Separate 1-way ANOVA were used. The production methods were used as factors, except for the comparisons between pre- and post-cryopreservation, where 2-way ANOVA were used. The level of significance was set at P≤0.05. A significant difference in re-expansion rates was found only at 24h post-thawing, with significantly higher rates in blastocysts produced in vitro compared to embryos of the Days 2-7 group. Levels ofmRNA expression were assessed using RT-qPCR. Before cryopreservation of embryos, no significant inter-group differences were seen. However, significantly more desmocollin 2mRNA expression was detected in embryos of the MOET group compared with blastocysts derived by the other production methods. Post-cryopreservation, blastocysts of 3 embryo production groups (IVP, MOET, Days 2-7) were available for analysis. Compared with levels ofmRNA expression before cryopreservation, re-expanded blastocysts after cryopreservation showed a significant up-regulation of heat shock protein A1A transcripts in all groups, and of solute carrier family 2 member 3 transcripts only in the IVP-derived group. The BAX, BCL-xL, occludin, and desmocollin 2 were significantly up-regulated in embryos of the MOET and IVP groups after cryopreservation, as compared with their counterparts before cryopreservation. None of the culture groups showed any pre- v. post-cryopreservation differences in the aquaporin 3 and the DNA methyltransferase 1mRNA levels. Blastocysts derived by transfer of in vitro derived cleaved stage embryos into the oviduct of synchronised heifers (Days 2-7) did not show any pre- v. post-cryopreservation differences in themRNA levels of any of the assessed genes. These results merit further investigation. After the process of cryopreservation and thawing, re-expanded embryos of the MOET and IVP groups do increase theirmRNA levels to prepare for hatching and further development.
Reproduction Fertility and Development 01/2011; 23(1):143-144. · 2.58 Impact Factor