In vitro production of bovine embryos in medium supplemented with a serum replacer: effects on blastocyst development, cryotolerance and survival to term

In this study, we evaluated a serum replacer (SR; Knockout SR, Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment 1 compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P<0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM+SR or (4) KSOM+FBS and cultured to Day 7 (fertilization=Day 0). Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P<0.05). Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24h survival rates (70.6%) than all other treatments (P<0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean=38.8 kg+/-1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

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 culture system on survival rate of vitrified bovine embryos produced in vitro

This study was designed to evaluate the effect of in vitro culture system on bovine blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were allocated to three culture conditions: (I) Oviductal cells-SOF (OCM-SOF); (II) Oviductal cells-TCM (OCM-TCM); and (III) SOF for 8 days. There was no significant difference between blastocyst rates among groups.In Experiment 2, the IVP-blastocysts in three above culture conditions were vitrified within groups segregated according to age (Day 7 and 8) and blastocoelic cavity size (early and expanded blastocysts). A trend of higher survival rate was obtained in vitrified/warmed early blastocysts compared with expanded ones, so that the difference in OCM-TCM group was significant (P < 0.001). Higher survival and hatching rates (P < 0.001) were obtained in OCM-SOF and OCM-TCM groups (co-culture) compared with SOF group and the age of blastocyst had no effect on post-thaw survival and hatching rates. In Experiment 3, after staining of blastocysts, in fresh blastocysts the highest number of trophectoderm cells was observed in OCM-TCM group and the number of inner cell mass (ICM) was higher in co-culture groups than SOF group (P < 0.001). In vitrified/warmed blastocysts the number of ICM and trophectoderm cells in co-culture groups was higher than SOF group (P < 0.001) except for the ICM of expanded blastocysts. In conclusion, in our culture conditions, the blastocyst yield is not influenced by culture system, while the cryotolerance of IVP-blastocysts is positively influenced by the presence of somatic cells. Moreover, the expanded blastocysts are more susceptible to cryoinjury than early blastocysts.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Administration of recombinant bovine somatotropin (rbST) at the time of breeding in superovulated fertile and subfertile ewes

Two experiments were conducted to evaluate the effects of the administration of 125 mg rbST at the time of breeding on fertile and subfertile superovulated ewes – in terms of the ovulation rate, embryo yield and embryonic development. In addition, the effects of the treatment on the plasma progesterone concentrations and interferon-tau (IFNT) expression in the embryos were evaluated in the fertile animals. In the first experiment, carried out on fertile superovulated ewes, the treatment significantly increased the ovulation rate (p < 0.05) and shifted the embryo distribution towards more advanced stages of development (p < 0.01). Plasma progesterone concentrations increased faster in the treated ewes, being significantly different (p < 0.05) between groups at day 6 post-breeding. In the second experiment, embryonic development was more advanced in fertile, but not in subfertile superovulated ewes. The expression of IFNT was significantly (p < 0.01) decreased in blastocyst embryos obtained from rbST treated animals, compared with non-treated ewes. It is concluded that the administration of rbST to subfertile ewes at the time of breeding does not stimulate embryonic development as it does when administered to fertile animals.     

Optimization of the processing of porcine platelet-rich plasma and its application on human mesenchymal stem cell cultivation

Because of ethical and scientific controversy, the utilization of fetal bovine serum (FBS) for cell culture medium must be minimized. This study develops porcine platelet-rich plasma (P-PRP) as a FBS substitute for human mesenchymal stem cell (hMSC) cultivation. Concentrating porcine blood by serial centrifugation to obtain P-PRP leads to activation by different agonist combinations to stimulate the secretion of growth factors. The concentration of growth factor in P-PRP is significantly increased by activation (p < 0.05). The concentration of PDGF, KGF and TGF-β in activated P-PRP is significantly higher than that in FBS. Design-expert was used to decide whether Co−T+Ca−, Co+T−Ca−, and Co+T+Ca− are optimal agonist formulations. MSC cultivation shows that the attachment rate, proliferation rate and viability of P-PRP supplemented media are significantly higher than those for FBS-supplemented and commercial media (p < 0.05). The results demonstrate that P-PRP is an optimal FBS substitute that supports in vitro h-MSCs expansion for subsequent biomedical applications.     

In vitro survival and development of goat preantral follicles in two different oxygen tensions

The aim of the present study was to evaluate the effect of two different oxygen (O₂) concentrations on survival and development of preantral follicles of goats cultured in vitro. Preantral ovarian follicles (≥150 μm) were isolated from ovarian cortex fragments of goats and individually cultured for 30 days under two different O₂ concentrations (5% and 20% O₂). Follicle development was evaluated on the basis of antral cavity formation, increase in follicular diameter, presence of healthy cumulus oocyte complexes and fully grown oocytes. Results showed with progression of culture period from 6 to 12 days, a decrease in follicular survival was observed in both O₂ concentrations (P < 0.05). When the O₂ tensions were compared to each other in the different days of culture, 20% O₂ was more efficient in promoting an increase in follicular diameter from day 24 of culture onward than 5% O₂ (P < 0.05). However, follicles cultured with 5% O₂ had an increased percentage of antrum formation from 12 days to the end of culture, compared with 20% O₂ (P < 0.05). Moreover, there was no difference in percentage of fully developed oocytes with the different O₂ tensions. However, only oocytes (16.7%) from follicles cultured in 20% O₂ resumed meiosis. In conclusion, concentration of 20% O₂ was more efficient in promoting follicular growth and oocyte meiosis resumption from preantral follicles of goats when grown in vitro.     

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P < 0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P > 0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P < 0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P > 0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.     

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus–oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0 mM, 0.02 mM, 0.2 mM, 2 mM, or 20 mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) at 38.7 °C in 7% CO₂ in air for 40–44 h. No significant difference (P > 0.05) in nuclear maturation was found between oocytes matured in TCM + pFF or PPM with 0 mM, 0.02 mM and 0.2 mM ammonium chloride. However, nuclear maturation was decreased (P < 0.05) in oocytes matured in PPM with 2 mM or 20 mM ammonium. After IVF, oocytes matured in PPM with 20 mM ammonium resulted in embryos with reduced (P < 0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20 mM ammonium resulted in embryos with lesser (P < 0.05) embryonic cleavage compared to TCM + pFF. However, PA embryos derived from oocytes matured in PPM with both 2 mM and 20 mM ammonium had reduced (P < 0.05) blastocyst development compared with TCM + pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.     

Influence of oocyte donor on in vitro embryo production in buffalo

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n = 40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A + B COC recorded in each 4-PS period had great repeatability (r = 0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A + B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r = 0.60; P < 0.001), SFL (r = 0.68; P < 0.001), COC (r = 0.48; P < 0.01) and Grade A + B COC (r = 0.40; P < 0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P < 0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r = 0.80; P < 0.05) and COC (r = 0.76; P < 0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.     

Effect of protein supplementation in potassium simplex optimization medium on preimplantation development of bovine non-transgenic and transgenic cloned embryos

The present study evaluated the effect of protein supplementation in potassium simplex optimization medium (KSOM) on bovine preimplantation embryo development. The in vitro fertilized (IVF) (Experiment 1), non-transgenic (Experiment 2) and transgenic cloned embryos (Experiment 3) were cultured for 192 h in KSOM supplemented with 0.8% BSA (KSOM-BSA), 10% FBS (KSOM-FBS) or 0.01% PVA (KSOM-PVA). Transfected cumulus cells with an expression plasmid for human alpha1-antitrypsin gene and a green fluorescent protein (GFP) marker were used to produce transgenic cloned embryos. Modified synthetic oviductal fluid (mSOF) supplemented with 0.8% BSA (mSOF-BSA) was used as a control medium. In Experiment 1, cleavage rate was significantly (P < 0.05) lower (69.1%) in IVF embryos cultured in KSOM-FBS than in KSOM-BSA (80.3%). The rate of hatching/hatched blastocyst formation was significantly (P < 0.05) lower in embryos cultured in KSOM-PVA than in KSOM-FBS (2.2% versus 10.8%). Blastocysts cultured in KSOM-FBS contained significantly (P < 0.06) higher numbers of inner cell mass cells (50.4 +/- 20.2) than those cultured in mSOF-BSA (36.9 +/- 19.2). In Experiment 2, the rate of blastocyst formation was significantly (P < 0.05) lower (20.5%) in embryos cultured in KSOM-PVA than in other culture media (33.3-38.5%). The rate of hatching/hatched blastocysts was significantly (P < 0.05) lower in KSOM-PVA (13.9%) and KSOM-FBS (17.1%) than in KSOM-BSA (30.8%) and mSOF-BSA (33.9%). The numbers of total and trophectoderm cells (104.6 +/- 32.2 and 71.7 +/- 25.5, respectively) were significantly (P < 0.05) lower in blastocysts cultured in KSOM-PVA than in KSOM-BSA (125.7 +/- 39.7 and 91.7 +/- 36.2, respectively). In Experiment 3, no significant differences in embryo development, GFP expression and blastocyst cell numbers were observed among the culture groups. In conclusion, the present study demonstrated that KSOM and mSOF supplemented with BSA were equally effective in supporting development of bovine non-transgenic and transgenic cloned embryos. Moreover, different developmental competence in response to protein supplementation of KSOM was observed between bovine non-transgenic and transgenic cloned embryos.     

Quantification and identification of fungal propagules in well-managed baled grass silage and in normal on-farm produced bales

The viable fungal spora of baled grass silage was quantified and identified. One group of bales (Experiment 1, n = 15) was wrapped in 6 layers of polythene, and was handled and stored for 6 weeks under well-managed conditions. This silage was free of visible fungal contamination after 6 weeks and the film surrounding these bales was visibly undamaged. Moulds were cultured from 9/15 bales, while yeasts were cultured from all bales. A second group of bales (Experiment 2, n = 18) from 9 farms, were wrapped in 4 layers of film, handled and stored for 8 months using normal on-farm procedures. Visible fungal contamination was observed on the surface of most of these bales (15/18) and the film surrounding some bales (8/18) was damaged. In silage sampled from parts of bales that were visually non-mouldy, yeasts were cultured from all bales and moulds from 15/18 bales. Bales in the well-managed group (Experiment 1) had numerically lower numbers of yeasts (mean: 9.7 × 10³ colony-forming units per gram of silage, cfu/g) and lower numbers of moulds (<10¹ cfu/g) compared to the normal on-farm produced group (2.3 × 10⁵ yeast and 1.5 × 10⁵ mould cfu/g; Experiment 2). The most common yeasts in each group of bales were Saccharomyces exiguus (12/15 bales; Experiment 1) and Pichia fermentans (11/18 bales; Experiment 2) and their numbers in all bales ranged from 0 to 10⁵ cfu/g (mean: 8.4 × 10³) and 0 to 1.5 × 10⁶ cfu/g (mean: 1.2 × 10⁵), respectively. Bales contaminated with visible mould growth on their surfaces had higher yeast and mould numbers in visually non-mouldy parts adjacent to the contaminated areas than bales that had no visible mould. Mould numbers were higher (P<0.05) in bales where the polythene film was visibly damaged compared to bales where the polythene film appeared intact. Penicillium roqueforti was not cultured from the well-managed bales, but it was the most common mould in bales prepared using normal on-farm procedures (13/18 bales); propagule numbers in bales ranged from 0 to 7.1 × 10⁵ (mean: 1 × 10⁵ cfu/g). Low numbers of mould propagules, the absence of viable P. roqueforti spores and the absence of mould growth in well-managed bales, emphasises the benefit of applying sufficient film and preventing it from becoming damaged during bale handling and storage.     

Assessment of the reproductive parameters,laparoscopic oocyte recovery and the first embryos produced in vitro from endangered Canindé goats (Capra hircus)

The Canindé breed of goats (Capra hircus) is currently endangered. The aims of this study were to characterize the estrus behavior, ovulatory responses and progesterone profiles, and to evaluate the in vitro embryo production (IVP) in this breed. In Experiment 1, ten nulliparous and seven pluriparous females received medroxyprogesterone acetate (MAP)-containing sponges (60 mg) plus 75 μg d-cloprostenol for estrus synchronization and their reproductive parameters were evaluated. In Experiment 2, oocytes obtained by laparascopy from hormonally stimulated females (n = 15) were used for IVP. There was no difference (p > 0.05) between nulliparous and pluriparous goats in terms of estrus response (40.0% vs. 85.7%), time from progestagen sponge removal to the onset of estrus (62.0 ± 15.5 vs. 50.7 ± 19.2 h; mean ± SEM), duration of estrus (25.0 ± 16.1 vs. 30.0 ± 15.1 h), percentage of ovulating animals (60.0% vs. 85.7%), number of ovulations (1.2 ± 0.4 vs. 1.3 ± 0.8), and diameter of the preovulatory follicle (5.8 ± 0.5 vs. 6.1 ± 0.3 mm). Progesterone concentrations were also similar (p > 0.05) in both groups. During laparoscopic recovery, there were average 12.2 aspirated follicles and 9.1 oocytes per goat, resulting in a high recovery rate (74.3%, 182/245). A total of 78 embryos were produced (51.0%). The mean number of cells in the blastocysts at day 7 of in vitro culture was 170.3 ± 12.5. In conclusion, nulliparous and pluriparous Canindé goats exhibited similar reproductive profiles. It was possible to produce embryos in vitro, allowing the instigation of an embryo bank for preservation of this breed.     

The effects of artificial activation timing on the development of SCNT-derived embryos and newborn piglets

This study investigated the effects of two different activation regimens on the developmental potential of somatic cell nuclear transfer (SCNT) embryos and postnatal survivability of the cloned piglets. In vitro matured oocytes were enucleated and reconstructed with porcine fetal fibroblasts. On the basis of the activation regimen used, the reconstructed porcine embryos were allocated into two groups: Group 1—simultaneous electrical pulses and activation group (SFA group); and Group 2—electrical fusion without calcium followed by electrical pulses with calcium after colcemid and cytochalasin B treatment for 5 h (DA group). Embryonic development in both SFA and DA groups was determined at day 6 of culture in NSCU-23 medium. To investigate the post-implantation development after the two activation methods, embryos were cultured for 1 day and then transferred into the oviducts of estrus-synchronized recipients. DA group had significantly (p < 0.05) higher cleavage rates than SFA group. However, the developmental rate to the blastocyst stage and the mean cell number of blastocysts did not differ (p > 0.05) between SFA and DA groups. Moreover, the pregnancy rate of SFA group was not significantly different compared to DA group. A total of 20 cloned piglets (SFA group-8 live piglets, DA group-11 live piglets and one stillborn) were obtained in the present study. The birth weight of the cloned piglets (live births) did not differ (p > 0.05) between the two groups. Furthermore, no difference was observed in the postnatal survival rates of the cloned piglets obtained using two different activation regimens. These results suggest that the timing of artificial activation and additional chemical treatments do not affect the developmental rate of porcine SCNT embryos. Remarkably, the pregnancy rate and postnatal survivability of the cloned piglets did not vary between SFA and DA groups.     

Effect of high tannin grain sorghum on gastrointestinal parasite fecal egg counts in goats

The objective of three experiments was to determine the influence of high condensed tannin (CT) grain sorghum on gastrointestinal parasite fecal egg counts (FEC). Sixteen naturally infected Boer crossbred mixed-sex goats were used. Animals that were supplemented with grain daily were removed from pasture and placed in individual pens and fed treatment diets for 21 or 14 days (Experiment 3). Goats were allowed ad libitum access to water and diets containing high or low CT cracked grain sorghum. On day 0 and every 7 days thereafter, PCV, FEC and FAMACHA^® eyelid color scores (EYE; Experiment 1 only) were recorded. For Experiment 1, percentage of animals dewormed, PCV and EYE were not influenced by treatment and averaged 13.6 ± 4.5% per treatment period, 23.4 ± 0.8% and 3.2 ± 0.12, respectively, for all animals. The FEC increased after day 0 for control but not high tannin grain sorghum fed goats (treatment by day interaction, P < 0.02). As expected, EYE and PCV were negatively related (r = −0.45; P < 0.0005). For Experiment 2, there was no influence of diet on PCV or FEC, but there was an effect of day on FEC (P < 0.002) in which all goats had higher FEC on day 0 (1956 ± 219 eggs/g) than any other sampling day. For Experiment 3, there was no effect of feeding high CT grain sorghum on FEC (2992 ± 591 eggs/g) or PCV (25.1 ± 0.5%). In these studies, high CT grain sorghum did not consistently influence FEC and did not impact PCV or number of animals requiring chemical anthelmintic treatment.     

Incorporating sheep into dryland grain production systems: I. Impact on over-wintering larva populations of wheat stem sawfly,Cephus cinctus Norton (Hymenoptera: Cephidae)

Wheat stem sawfly (WSS), Cephus cinctus Norton (Hymenoptera: Cephidae) is the most damaging insect pest to Montana's $ 1 billion dollar per year grain industry. Current WSS control methods are either expensive, reduce wheat yields, or are not effective. Our objective was to compare burning, grazing, tilling, trampling and clipping wheat stubble fields on over-wintering WSS larval populations. Treatments were evaluated in three experiments using a randomized complete block design and four replications at each site. Eight, six, and two sites were used for Experiments 1, 2, and 3, respectively. Contrast statements were used to make pre-planned comparisons among treatments. For Experiment 1, treatments were fall tilled, fall grazed, spring grazed, fall and spring combined (Fall/Spr) grazed, and an untreated control. Five mature ewes were confined with electric fence to 111 m² plot for 24 h in the fall and spring grazed treatments resulting in a stocking rate of 452 sheep d/ha. For Fall/Spr, the stocking rate was 904 sheep d/ha. For Experiment 2, treatments were fall grazed, fall burned, fall tilled, and an untreated control. In Experiment 3, treatments were fall trampled, spring trampled, Fall/Spr trampled, hand clipped to a stubble height of 4.5 cm, and an untreated control. Trampled treatments were done at the same stocking rates as grazing treatments but sheep were muzzled to prevent intake. Wheat stem sawfly larval numbers were collected in the fall and spring, pre- and post-treatment, respectively, by collecting all plant material from three, 0.46 m lengths of row and counting the number of live larvae present. In Experiment 1, WSS mortality was greater (P < 0.01) for the mean of all grazed treatments (68.4%) than either control (43%) or tilled (47%) plots. Mortality did not differ (P = 0.75) between fall (67%) and spring (64%) grazed plots but was greater (P = 0.02) for Fall/Spr (74%). In Experiment 2, larva mortality was greater (P < 0.01) for fall grazed (63%) than burned plots (52%). In Experiment 3, WSS mortality was greater (P < 0.01) for the mean of all trampling treatments (57%) than either control (33%) or clipped (32%) plots. Mortality did not differ (P > 0.25) between fall (54%) and spring trampling (47%) but was greater (P = 0.01) for Fall/Spr (70.6%). No differences (P > 0.25) were detected for WSS mortality when grazing was compared to trampling. These results indicate the potential for using grazing sheep to control wheat stem sawfly infestations in cereal grain production systems.     

Effect of follicular wave synchronization on in vitro embryo production in heifers

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 7 d, followed by the administration of 150 μg d-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. 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 28 d. Heifers from Group 1X-EB also received 2 mg 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 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. 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.