Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls

The objective of this study was to determine the effect of staining bovine sperm, with or without flow cytometry, on in vitro fertilization of bovine oocytes and blastocyst development. Bovine oocytes (n=4273) were fertilized with frozen–thawed sperm from three bulls that was: stained with Hoechst 33342 and sorted (into X- or Y-chromosome-bearing sperm) with flow cytometry; stained but not sorted; and not stained or sorted (Control). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 (supplemented with 10% fetal calf serum and 15 ng FSH, 1.0 μg LH, 1.0 μg E2/ml) for 22–24 h at 39 °C in 5% CO₂ in air with maximum humidity. Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1) 6–7 h after insemination and cultured for 65–66 h. Embryos that had cleaved by 72 h post-insemination were cultured an additional 96 h in CDM-2 containing 0.12 IU insulin/ml. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7–8 after insemination, respectively. There was no significant difference in blastocyst rate among the three types of sperm; however, cleavage rates with stained and sorted sperm (53.1%) and unsorted, stained sperm (59.9%) were lower (P<0.05) than Control sperm (69.7%). Furthermore, there were significant differences due to semen from different bulls in cleavage and blastocyst rates.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% approximately 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 muM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% approximately 57.3%; 22.3% vs 7.4% approximately 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% approximately 46.2%; 18.0% vs 7.1% approximately 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8-74.4% vs 56.5-57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% approximately 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3-27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.     

Development and calcium level changes in pre-implantation porcine nuclear transfer embryos activated with 6-DMAP after fusion

This study investigated the effect of treatment with 6-dimethylaminopurine (6-DMAP) following fusion on in vitro development of porcine nuclear transfer (NT) embryos. Frozen thawed ear skin cells were transferred into the perivitelline space of enucleated oocytes. Reconstructed oocytes were fused and activated with electric pulse in 0.3 M mannitol supplemented with either 0.1 or 1.0 mM CaCl(2). In each calcium concentration, activated oocytes were divided into three groups. Two groups of them were exposed to either ionomycin (I + 6-DMAP or 6-DMAP alone. In experiment 2, fused NT embryos in 0.3 M mannitol containing 1.0 mM CaCl(2) were exposed to 6-DMAP either immediately or 20 min after fusion/activation. For 0.1 mM CaCl(2), oocytes activated with either I + 6-DMAP or 6-DMAP alone showed a higher (P < 0.05) developmental rate to the blastocyst stage than those activated with an electric pulse alone (26.7 and 22.5 vs. 12.5%). For 1.0 mM CaCl(2), oocytes activated with either I + 6-DMAP or 6-DMAP alone showed significantly higher (P < 0.05) developmental rate to the blastocyst stage (35.6 and 28.3 vs. 19.8%). Developmental rate to the blastocyst stage was (P < 0.05) increased in NT embryos activated with 6-DMAP 20 min after fusion. 6-DMAP made a higher and wider Ca(2+) transient compared to that induced by electric pulses (Fig. 3). The fluctuation lasted during the time that oocytes were cultured in 6-DMAP. Regardless of Ca(2+) concentration in fusion medium, activation with 6-DMAP following electric pulses supported more development of porcine NT embryos. Activation of NT embryos with 6-DMAP after fusion in the presence of 1.0 mM CaCl(2) could support better developmental rate to the blastocyst stage.     

Effects of floor-feeding and the presence of a foraging substrate on the behaviour and stress physiological response of individually housed gilts

Both restricted feeding and barren housing have a negative influence on sow welfare. The aim of this study was to test whether sows that have to search for their feed in a substrate on the floor show less stereotyped (and other abnormal) behaviour and have a lower physiological stress response. In three batches, 96 gilts were housed individually in two rooms in 3.1 m² pens with 1.9 m² solid floor. In a 2×2 factorial design either wood shavings (S) or no substrate (NS) were provided on the floor, and 900 g of feed was provided twice daily (06:30 and 15:00 h) either in a trough (T) or on the floor (F). In weeks 8–12, behaviour was scan-sampled once in the periods 07:00–09:00 h (P1), 10:00–12:00 h (P2), and 13:00–15:00 h (P3). Data from the 5 weeks were pooled per animal. Video recordings (24 h) in week 12 or 13 were scan-sampled for ‘standing’. Saliva samples were taken in week 11 at 2 h intervals during 24 h and measured for cortisol. Spontaneously voided morning-urine was sampled in weeks 2, 7 and 12 or 13 for determination of ratios of adrenaline (A) and noradrenaline (NA) with creatinine (CR). Most effects that were found were due to substrate presence. Main findings were that compared with S-animals, NS-animals stood more during the dark period (4.8% versus 3.0%; P<0.05) and showed more (visible) oral behaviour in P1 (56.8% versus 47.6%; P<0.05), P2 (35.8% versus 30.8%; tendency) and P3 (44.1% versus 33.8%; P<0.05). This included more sham chewing in P1 (tendency), P2 and P3, more pen manipulation in P3, and more other oral behaviour (e.g. teeth grinding) in P1 (tendency) and P2. They had higher cortisol levels before feeding (peaks) and in the early evening (24 h average: 1.49 ng/ml versus 1.02 ng/ml; P<0.05) and higher NA/CR ratios in weeks 7 (tendency) and 12 (5.5 ng/mg versus 3.7 ng/mg; P<0.05). No treatment effects were found on A/CR ratios. Substantial interactive effects of feeding method and substrate were only found in floor manipulation. In all three periods NS-T-animals manipulated the floor less than other animals, probably because they had no attraction to the floor and were not rewarded for searching. Results imply that the presence of a substrate on the floor improves welfare, whereas provision of feed in the substrate in order to stimulate foraging behaviour is of less importance.     

Effects of different activation protocols on preimplantation development, apoptosis and ploidy of bovine parthenogenetic embryos

The objective of this study was to optimize the protocols for bovine oocytes activation through comparing the effectiveness of different treatments on the activation and subsequent development of oocytes and examining the effects of two combined activation treatments on the blastocyst apoptosis and ploidy. Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation, cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage. In Experiment 1, the matured oocytes were treated with single activation agents, including ionomycin (5 microM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5 microM for 5 min) or strontium (10mM for 5h). The pronuclear formation and cleavage rate were higher significantly in ionomycin (39.0 and 30.7%) and ethanol (41.5 and 28.1%) treatment alone compared to other treatments (9.7-25.2 and 11.3-23.7%, respectively, P<0.05). Very low blastocyst rates (3.9-5.3%) resulted which were not significantly different among treatments (P>0.05). For the combined activation treatment (Experiment 2), the same concentrations of ionomycin and ethanol as in Experiment 1 were used in combination with either 6-dimethylaminopurine (6-DMAP, 2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10 microg/ml for 3 h). The pronuclear formation, cleavage rate, blastocyst rate and cell number of blastocyst were higher significantly (P<0.05) in ionomycin+6-DMAP treatment (67.1, 69.2, 28.0 and 91.3%, respectively) and ethanol+CHX+CB treatment (68.9, 70.2, 25.5 and 89.3%, respectively) compared to other treatments (11.7-58.1, 10.2-47.1, 1.5-24.2 and 34.2-62.7%, respectively). In Experiment 3, the parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis using a terminal deoxynucleotidyl transferase mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The ethanol+CHX+CB treatment (7.0%) showed significantly lower blastocyst apoptosis index compared to ionomycin+6-DAMP treatment (9.1%, P<0.05). Furthermore, the chromosomal composition in the parthenotes embryos differed (P<0.05) among treatments. The percentage of haploid parthenotes was higher in ionomycin+6-DMAP treatment than ethanol+CHX+CB treatment. These results suggested that ethanol+CHX+CB treatment was more favorable protocol for parthenogenesis of bovine oocytes.     

Oocyte activation procedures and influence of serum on porcine oocyte maturation and subsequent parthenogenetic and nuclear transfer embryo development

The viability of SCNT embryos is poor, with an extremely low cloned piglet production rate. In the present work, we studied the effect of three activation protocols based on ionomycin treatment (5 microM ionomycin for 5 min and incubated in 2 mM 6-DMAP for 3.5 h) or electric stimuli (two square wave electrical DC pulses of 1.2 kV/cm for 30 micros) combined or not with 6-DMAP on parthenogenetic embryo development. Oocytes activated by ionomycin plus 6-DMAP showed lower cleavage (47.2 vs. 78.5-81.5; p < 0.05) and blastocyst rates (11.3 vs. 29.2-32.1; p < 0.05) than those activated by electrical and electrical plus 6-DMAP treatments. Also, we studied the effect of addition of serum to maturation medium (0% vs. 10%) on nuclear maturation and further parthenogenetic and SCNT embryo development. We observed in the parthenogenetic embryos that cleavage rates in the serum-free group were significantly higher than in the serum-supplemented group (81.8 vs. 69.6% respectively; p < 0.05), although these differences were not detected in blastocyst rates or blastocyst nuclei numbers. Regarding SCNT embryos, no significant differences were observed in cleavage or blastocyst rates between different experimental groups of SCNT embryos. In conclusion, electrical pulse followed or not by 6-DMAP was found to be an efficient procedure to artificially activate MII porcine oocytes. Moreover, the addition of serum to oocyte maturation media did not seem to improve parthenogenetic or SCNT porcine embryo development.     

Effects of oilseed meals and grain–urea supplements fed infrequently on digestion in sheep: 2. Cereal straw diets

An experiment examined the intake, growth responses and rumen digestion of young sheep fed ad libitum oat or barley straws alone or supplemented with approximately isonitrogenous amounts of barley grain and urea (Bar/N), safflower seed meal (SAF) or linseed meal (LIN) supplements provided at 3 day intervals. The supplements comprised 15–22% of total dry matter (DM) intake. Sheep offered either of the straws alone consumed 35.0–37.2 g DM/kg liveweight (LW^0.75) per day of straw and an estimated 2.03–2.07 MJ metabolizable energy (ME) per day, and lost 85–97 g LW per day. Supplements increased (P<0.05 or <0.001) voluntary intake of straw and of total DM, and the organic matter (OM) digestibility of the entire diet. Each of the supplements increased (P<0.001) the estimated ME intake to a similar extent and changed the rapid LW loss of sheep fed straw alone to approximate LW maintenance. Rumen ammonia concentrations in sheep fed barley and oat straws alone (12 and 24 mg NH₃/l, respectively) were expected to be deficient for microbial activity, but were increased (P<0.001) by provision of the supplements. Digestion of straw in synthetic fibre bags incubated in the rumen was markedly increased (P<0.01 or <0.001) when supplements were provided. Rumen pH was depressed briefly to pH <6.0 by the Bar/N, but not by the LIN or SAF, supplements. In young sheep fed cereal straws and losing LW rapidly the oilseed meal supplements increased wool growth more than the barley grain–urea supplements, but both types of supplement increased ME intake similarly and were equally effective to reduce the extent of LW loss.     

A note on the effect of gestation housing environment on approach test measures in gilts

Systems’ welfare evaluation, including behavioural testing, is becoming increasingly popular in farm animal assurance schemes. The aims of this study were to investigate whether fairly short-term exposure to gestation housing systems, which varied in physical, environmental and human-input factors, influenced behavioural and physiological measures during a human approach test—often used to identify problems in human–animal interactions. Twenty-four Large White×Landrace gilts were initially subjected to identical human contact and daily husbandry. Forty-two days after service, the gilts were randomly assigned to either an indoor housing system (n=16) or an outdoor housing system (n=8), which differed physically and in the amount of human contact and daily husbandry. The indoor system used an electronic sow feeder (ESF), was more space-limited and thermally-controlled and had human contact centered on cleaning out. The outdoor system was more extensive, had much greater space accessible, was not thermally-controlled and had human contact that centered around feeding. The human approach test was carried out on all gilts 30–44 days after entry to the gestation system. At testing, each individual was fitted with heart rate monitor and then moved into a test arena. After 2 min an unfamiliar human entered the pen and stood motionless for 3 min against one wall and then approached the gilt and touched her snout. Throughout the experimental period, behaviour and sound within the test arena were recorded continuously. During the 2 min familiarisation period, outdoor gilts had lower heart rates (108.2 bpm versus 123.7 bpm, P<0.05) and tended to perform fewer short vocalisations (0.5 calls per min versus 3.4 calls per min, P<0.1). Outdoor-housed gilts also carried out less locomotor behaviour (2.2 sections crossed versus 4.0 sections crossed, P<0.05) and tended to perform fewer short (1.4 calls per min versus 5.0 calls per min, P<0.1) and long vocalisations (0.2 calls per min versus 1.8 calls per min, P<0.1) over the 3 min test period. Outdoor gilts tended to be slower to approach within 0.5 m of the human (69.9 s versus 19.3 s, P<0.1) but they then took less extra time to make physical contact (3.3 s versus 52.7 s, P<0.1). Mean heart rate was significantly lower in outdoor sows over the whole 3 min period (99.5 bpm versus 115.5 bpm, P<0.05). The results demonstrate that short-term exposure to different housing systems did influence behavioural and physiological measures during a standard human approach test and thus, systems differences should be taken into account before making judgements about the human–animal relationship on any commercial farm, based on results of behavioural tests of this type.     

Effects of expander-treating a barley-based concentrate on ruminal fermentation, bacterial N synthesis, escape of dietary N, and performance of dairy cows

Three primiparous dairy cows in early lactation with cannulas in rumen, duodenum and ileum were used in a 3×3 Latin square design to study effects of expander treatment of a barley-based concentrate. The concentrate was either pelleted at 75–80°C or expander treated at 125–130°C prior to pelleting. The diets consisted of 6.7 kg DM of grass silage and 10 kg DM of (1) 100% pelleted, (2) 50% pelleted and 50% expanded or (3) 100% expanded concentrate. The diets were offered as a mixed ration in four equal meals daily. Ruminal fermentation, bacterial N synthesis, duodenal, ileal and faecal flow of nutrients, and animal performance were monitored. Expander treatment numerically increased ruminal digestion of starch, which explained the observed increase in ruminal VFA concentration and the lowered ruminal pH (P<0.05). The proportion of butyrate in rumen liquid increased, whereas the proportion of propionate decreased in the expanded compared to the pelleted treatment (P<0.05). Expander treatment tended to increase rumen volume and rumen NDF pool size. Ruminal digestion of NDF was numerically lower in the expanded than in the pelleted treatment. No differences in bacterial N synthesis or efficiency of synthesis were observed among treatments. Expander treatment numerically increased the duodenal flow of non-ammonia N (NAN) and amino acid N (AAN), and seemed to increase the flow of non-ammonia non-bacterial N (NANBN) to the duodenum to a similar extent as was indicated by nylon bag studies. Milk production and milk fat and protein content were increased by the expander treatment (P<0.05), indicating that expander treatment increased the supply of nutrients for milk production.     

The effect of follicle-stimulating hormone on follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor-I in the goat ovary

The effect of FSH on goat follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor (IGF)-I were studied through both in vivo and in vitro experiments. The FSH treatment was begun on Day 9 after estrus and consisted of injections twice a day for 3 days in decreasing doses (7.5–7.5–5.0–5.0–2.5–2.5 mg). Does in both treatment and control groups were slaughtered for ovaries on Day 12. Granulosa cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Expression of IGF-I and IGF-II mRNA was determined by RT–PCR, while concentrations of progesterone (P4), estradiol (E2), IGF-I and IGF-II were measured by radioimmunoassay (RIA). Following parameters increased significantly (P<0.05) after the FSH treatment: follicle number (5.0±1.5 versus 9.0±2.0 per ovary), the level of E2 (0.1±0.1 ng/ml versus 0.7±0.2 ng/ml), the E2/P4 ratio (0.7±0.4 versus 4.7±3.0) and the concentrations of IGF-I (0.5±0.2 ng/ml versus 119.4±15.1 ng/ml) and IGF-II (0.12±0.03 ng/ml versus 40.9±18.7 ng/ml) in follicular fluid of the medium sized (3–5 mm) follicles and in the ovarian cortex the relative quantity of IGF-I mRNA (0.37±0.17 versus 0.90±0.12 Max OD). In contrast, the ratio of apoptotic granulosa cells in these follicles was reduced significantly (0.53±0.1 versus 0.10±0.01, P<0.05). In large (>5 mm) follicles, however, only the follicle number (2.3±0.7 versus 7.0±1.5 per ovary) and the level of IGF-I (38.4±11.0 ng/ml versus 87.3±13.9 ng/ml) increased significantly (P<0.05), whereas other values did not change. In vitro culture of granulosa cells showed that FSH significantly (P<0.05) enhanced IGF-I production (12.7±2.1 ng/ml versus 26.±21.9 ng/ml) by these cells, and both FSH and IGF-I reduced the ratios of apoptotic cells (from 0.7±0.07 to 0.3±0.1 and 0.2±0.04, respectively) and the effect was additive when both were used together. H89, the PKA pathway inhibitor, blocked the effect of FSH on granulosa cell apoptosis and IGF-I production in vitro. These results indicated that FSH mainly enhanced the development of medium sized follicles in the goat by suppressing the apoptosis of granulosa cells via increasing production of IGF-I and steroids, possibly through the PKA pathway.     

Optimisation of porcine oocyte activation following nuclear transfer

Experiments were conducted to examine the effects of (a) different activation methods, (b) incubation time in calcium-free medium and (c) bisbenzimide staining on the activation and subsequent development of pig oocytes. Oocytes were matured in vitro and activated by one of the following methods: combined thimerosal/dithiothreitol (DTT) treatment, calcium ionophore A23187 treatment followed by incubation in the presence of 6-dimethylaminopurine (6-DMAP), electroporation, and electroporation followed by incubation with cytochalasin B. There were no significant differences in the activation rate (ranging from 70.0% to 88.3%) and the percentage of cleaved embryos after activation (ranging between 48.8% and 58.8%) among the four treatment groups (p < 0.05). The rate of development of the blastocyst stage in oocytes activated by thimerosal/DTT (10.0%) or electroporation followed by cytochalasin B treatment (12.3%) was significantly higher (p < 0.05) than in the group activated with A23187/6-DMAP (2.5%). Both the activation rate and the rate of blastocyst formation in oocytes that were incubated in Ca(2+)-free medium for 8 h before thimerosal/DTT activation were significantly lower (p < 0.05) than in those incubated for 0, 1 or 4 h. Intracellular Ca2+ measurements revealed that the Ca2+ homeostasis in these oocytes were severely altered. Staining of oocytes with 5 micrograms/ml bisbenzimide for 2 h decreased the quality of blastocysts and increased the rate of degenerated embryos at day 6. Two activation protocols (thimerosal/DTT and electroproation) were used for activation after nuclear transfer; the rate of nuclear formation did not differ in the oocytes activated by the two different methods.     

Effect of ambient temperature on in vitro fertilization of Bubaline oocyte

The present study was used to examine the effect of ambient temperature on the day of slaughter of buffaloes on oocyte cleavage and subsequent embryo development following in vitro fertilization (IVF)/chemical activation (parthenogenesis). A total of 601 oocytes were collected from buffaloes, which were sacrificed when the ambient temperature was >40 or ≤40 °C and the collected oocytes were matured in vitro. During each experiment about half of the matured oocytes were used for IVF whereas the remaining oocytes were subjected to one of the three chemical activation protocols viz. (i) 7% ethanol (ET) and 6-di methyl amino purine (6-DMAP), (ii) ET and cycloheximide (CHX) and (iii) ET followed by a combined treatment of 6-DMAP and CHX. Cleaved oocytes were cultured in mSOF supplemented with BSA, essential amino acids, non-essential amino acids, ITS (insulin transferrin and selenium) and l-glutamine. Low cleavage and subsequent embryo development was observed in those oocytes which were collected from buffaloes slaughtered at ambient temperature >40 °C than at ≤40 °C. There was no significant difference in cleavage rate following different chemical activations in oocytes collected from buffaloes slaughtered on the day when the maximum ambient temperature was >40 °C or ≤40 °C. These results suggest that high ambient temperature influences competence of oocytes to cleave and develop to blastocyst stage following natural activation with sperm and/or process of fertilization and subsequent embryo development.     

A method to reduce the invasiveness of fetal catheterization in the cow

Surgical intervention in general anesthesia (GA) of the cow in late gestation is a stressful condition for both mother and fetus, potentially leading to premature delivery or fetal death. The present study hypothesized that fetal catheterization at days 246–253 (90% of gestation) is done with less physical and metabolic stress for the mother and fetus, when the surgery is performed on a standing cow and local anesthesia (LA) rather than on a recumbent cow in general anesthesia. Fetal and uterine maternal intra-vascular catheters were implanted during general anesthesia (GA, n=24) or local analgesia (LA, n=7). Blood gases and metabolite levels in the fetal calves and their mothers were measured during surgery and for 5 days post-operatively. During surgery, venous blood pH was higher (7.44±0.01 versus 7.39±0.01, P<0.05) and hemoglobin and oxygen contents lower in LA cows compared with GA cows (9.3±0.3 mg/dl versus 11.8±0.2 mg/dl, P<0.001 and 10.1±0.3 ml/dl versus 12.6±0.6 ml/dl, P<0.05). The differences between the two groups of fetuses reflected those of their dams in that LA fetuses showed lower arterial oxygen pressure (18.3±1.4 mmHg versus 24.8±1.4 mmHg, P<0.05) and hemoglobin (7.81±0.30 mg/dl versus 9.22±0.21 mg/dl P<0.01) and furthermore, they also showed higher blood glucose (2.4±0.2 mM versus 1.4±0.1 mM, P<0.01). During the 5 days post-surgery, 10 GA fetuses (42%) and 1 LA fetus (14%) died in utero. Bacterial contamination was implicated in six of the GA deaths and in the one LA death. In the dams with surviving calves, differences in hemoglobin (9.49±0.21 mg/dl versus 11.17±0.23 mg/dl P<0.001) and O₂ct (10.9±0.3 ml/dl versus 12.5±0.5 ml/dl, P<0.05) were still present, and in addition, blood glucose was higher in LA versus GA cows (4.3±0.2 mM versus 3.8±0.1 mM, P<0.05). The choice of surgical method did not affect post-surgery blood chemistry in the surviving fetuses, except that the higher blood glucose in the LA fetuses at surgery tended to be maintained also post-operatively (2.0±0.2 mM versus 1.5±0.1 mM, P=0.07). The observed differences in blood chemistry parameters between the two methods of surgery and possibly in the fetal death may be explained by differences in catheterization method and the associated differences in physical and metabolic stress during and after surgery. Thus, surgery upon a standing cow in local anesthesia should be considered as an alternative to surgery in universal anesthesia for fetal catheterization in the cow in late gestation.     

Coincident increases in oxytocin receptor expression and EMG responsiveness to oxytocin in the ovine cervix at oestrus

This study has localised oxytocin receptor (OTR) mRNA expression within the cervix of non-pregnant ewes and related this to changes in the sensitivity of the cervical musculature to administered oxytocin (OT) during the oestrous cycle by recording electromyographic (EMG) activity. Cervices were collected from 34 ewes at specified time points throughout the cycle. OTR mRNA expression was localised by in situ hybridisation and results were expressed as optical density measurements from autoradiographs in each of four different cervical regions. EMG recordings were made for up to 8 h per day from four non-pregnant ewes undergoing seasonal oestrous cycles between Days −3 and +3 relative to oestrus (Day 0). The highest concentrations of OTR mRNA were detectable within the luminal epithelium (LE) of the cervix, with values increasing from Day 15 of the cycle, peaking during the follicular phase (P<0.001, compared to the mid-luteal phase) and returning to basal by Day 2. There was a small but significant increase in OTR mRNA hybridisation (above basal/luteal phase values) within the stromal cells (STR) adjacent to the lumen (P<0.05) during the same time period, but no differences from basal values were detectable in the dense collagenous annular ring or in tissue superficial to this. Analysis of pooled EMG activity recorded daily from the cervix indicated that endogenous contractile activity was higher on Day 0 than on the Days +1 (P<0.05), −2, +2 and +3 (P<0.001). The response to bolus intravenous (i.v.) injections of 25 mU OT (25 mU) varied with day of the cycle. This dose produced a measurable and significant response on Days 0 (P<0.001) and +1 (P<0.001), but not on any of the other days, indicating that the sensitivity of the cervical musculature to OT peaked on these days. These data show that the cervix is highly responsive to OT at oestrus. This coincides with an increase in OTR mRNA expression in the luminal epithelial cells, suggesting the likely production of an intermediary messenger between the epithelial and smooth muscle cells.     

Examination of cyclic changes in bovine luteal echotexture using computer-assisted statistical pattern recognition techniques

B-mode sonography is a well-established diagnostic tool for determination of cycle stage in gynaecology. The aim of this study was to determine whether computer-assisted texture analysis of B- mode sonographic images of bovine luteal glands provides further information about the animal's plasma progesterone concentration and cycle stage. Four Simmenthal cows were examined during two consecutive estrous cycles with an ultrasound device equipped with a 7.5 MHz microconvex probe. During each examination three B-mode images of the corpus luteum (CL) were digitized and analyzed off-line using a computer-assisted texture analysis program. Size, echogeneity, and echotexture of the CL were characterized by the following texture parameters: area of cross-sectional planes of the CL (A), mean gray level (MGL), correlation (CORR), run percentage (RPERC), and long-run emphasis (LREM). Plasma progesterone levels (P4) were also determined. All parameters showed characteristic changes during the estrous cycle (P < 0.05). Variance component estimates for the effect of Day of estrous cycle on A, MGL, CORR, RPERC, and LREM were 56.6%, 64.6%, 77.6%, 89.9%, and 86.0%, respectively, and 20.6%, 24.5%, 7.2%, 0.0%, and 14.0% for the influence of the individual cow. The factor estrous cycle within cows was responsible for 22.8%, 10.9%, 15.2%, 10.1%, and 0.0% of the variability of A, MGL, CORR, RPERC and LREM values, respectively. Cyclic changes were similar in A and P4. In contrast to P4, which decreased already between Days –5 and –3 (Day 0 = ovulation), A stayed at constant high values until Day –3. Mean MGL values were higher (P < 0.05) on Days 7, 9, and 13 compared to Days 3 and –3. Mean CORR values were constantly high (P > 0.05) during the first days after ovulation and decreased continuously (P < 0.05) between Days 5 and 13. Thereafter, mean CORR values remained low (P < 0.05) until the next ovulation, except on Day –3 (P < 0.05). Mean RPERC rose between Days 1 and 9 from low to high values (P < 0.0001) remained at these high values (P > 0.05) between Days 9 and 15, and decreased (P < 0.05) afterwards to baseline values on Day –1. Mean LREM inclined steeply (P < 0.0001) from minimum to maximum between Days 1 and 5. From Days 7 to –3, mean LREM remained (P > 0.05) at a constant level close below the maximum value, and decreased to baseline values on Day –1. The results of this study show that statistical pattern recognition techniques provide new information about the luteal glands, thus facilitating a more accurate differentiation between different cycle stages in cows.     

The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX- treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.     

Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p < 0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.     

Phytohemagglutinin improves efficiency of electrofusing mammary gland epithelial cells into oocytes in goats

The objective was to investigate the effect of phytohemagglutinin (PHA) on the fusion of mammary gland epithelial (MGE) cells into enucleated oocytes in goats. The toxicity of PHA was evaluated by testing its effect on the development of parthenogenetic caprine oocytes. The effective dose and duration of PHA treatment (100 μg/mL, 20 min incubation) was selected and used to compare fusion efficiency and embryo development following nuclear transfer. Two electrofusion protocols, chamber fusion (CF) and pressurized microelectrode fusion (pMEF), were also compared, when couplets were treated with and without PHA (100 μg/mL, 20 min). Fusion rate of couplets increased from 52.8 to 74.0% for the CF protocol (P < 0.05), but was not significantly different for the pMEF protocol (72.7% vs. 78.1%) after PHA treatment. There were no significant differences between treated group and control in rates of subsequent cleavage or blastocyst development. Following transfer of the cloned blastocysts derived from the PHA-treated group and the control group into synchronized recipients, pregnancy rates (Day 30) were not significantly different between treated group and control (28.6% vs. 25.0%). However, all recipients aborted within 120 d, microsatellite DNA analyses confirmed that the aborted fetuses were genetically identical to the donor goat. In conclusion, the fusion rate of caprine MGE cell couplets was improved by pre-incubating couplets in medium containing 100 μg/mL PHA prior to electrical pulsing, and embryos derived from PHA treatment established early pregnancies.     

Nitrogen metabolism and blood metabolites in three goat breeds fed increasing amounts of protein

Twelve wethers (27–33 kg and 12–14 months of age) representing meat (Nubian), milk (Alpine) and mohair (Angora) producing goats were used to evaluate breed differences in protein utilization with diets containing 9 (L), 15(M) or 21% CP (H) and 2.4 Mcal ME/kg DM in a digestion trial. Fecal N, urine N and absorbed N as percent of N intake were not affected (P>0.05) by breed. Retained N as percent of absorbed N was not different (P>0.05) between breeds. Ruminal propionate (molar %) was lower (P<0.05) in Angora, but other ruminal VFA and ammonia-N were not affected (P>0.05) by diet or breed. Plasma urea-N increased (P<0.01) with dietary CP level (8.3, 22.0 and 33.3 mg/dl for L, M and H, respectively), and concentrations were lowest for Angoras and highest for Nubians (18.5 vs. 21.2 vs. 23.9 mg/dl) (P<0.01). Plasma total protein (mean 69.7 g/l), glucose (mean 83.1 mg/dl), non-esterfied fatty acids (mean 101.4 μEq/l) and cortisol (mean 10.8 ng/ml) were not affected (P>0.05) by breed or diet. Plasma glucagon concentrations increased (P<0.05) with increasing CP intake (72.4, 167.6 and 239.1 pg/ml for L, M and H, respectively). The study indicated that there was no apparent breed difference between Nubian, Alpine and Angora goats in nitrogen utilization when goats were fed pelleted diets containing 9 to 21% CP.