Effects of increasing amounts of hempseed cake in the diet of dairy cows on the production and composition of milk

This study explored the potential for using seed cake from hemp (Cannabis sativa L.) as a protein feed for dairy cows. The aim was to evaluate the effects of increasing the proportion of hempseed cake (HC) in the diet on milk production and milk composition. Forty Swedish Red dairy cows were involved in a 5-week dose-response feeding trial. The cows were allocated randomly to one of four experimental diets containing on average 494 g/kg of grass silage and 506 g/kg of concentrate on a dry matter (DM) basis. Diets containing 0 g (HC0), 143 g (HC14), 233 g (HC23) or 318 g (HC32) HC/kg DM were achieved by replacing an increasing proportion of compound pellets with cold-pressed HC. Increasing the proportion of HC resulted in dietary crude protein (CP) concentrations ranging from 126 for HC0 to 195 g CP/kg DM for HC32. Further effects on the composition of the diet with increasing proportions of HC were higher fat and NDF and lower starch concentrations. There were no linear or quadratic effects on DM intake, but increasing the proportion of HC in the diet resulted in linear increases in fat and NDF intake, as well as CP intake (P < 0.001), and a linear decrease in starch intake (P < 0.001). The proportion of HC had significant quadratic effects on the yields of milk, energy-corrected milk (ECM) and milk protein, fat and lactose. The curvilinear response of all yield parameters indicated maximum production from cows fed diet HC14. Increasing the proportion of HC resulted in linear decreases in both milk protein and milk fat concentration (P = 0.005 and P = 0.017, respectively), a linear increase in milk urea (P < 0.001), and a linear decrease in CP efficiency (milk protein/CP intake; P < 0.001). In conclusion, the HC14 diet, corresponding to a dietary CP concentration of 157 g/kg DM, resulted in the maximum yields of milk and ECM by dairy cows in this study.     

Comparisons between nulliparous heifers and cows as oocyte donors for embryo production in vitro

The aims of the present study were to compare (1) Holstein-Friesian heifers versus early postpartum lactating cows, and (2) different age categories of crossbred beef heifers versus cows, in terms of oocyte yield, morphological quality and developmental competence. Four experiments were designed to test the associated hypotheses. In Experiment 1, ovum pick up was carried out twice weekly for a period of 5 weeks on Holstein-Friesian heifers (n = 8) and early postpartum cows (n = 8). Oocytes were submitted to in vitro maturation (IVM), fertilization and culture. Significantly more follicles were punctured on the ovaries of heifers than cows (10.4 versus 7.8, P < 0.001). This was reflected in a significantly higher number of total oocytes (4.7 versus 2.8, P < 0.001) and grade 1-2 oocytes recovered/animal from heifers than from cows (3.0 versus 1.8, P < 0.05). There was no significant difference in the percentage of oocytes cleaving after fertilization, or in the percentage reaching the blastocyst stage between heifers and cows. In Experiment 2, oocytes were obtained by manual aspiration from the ovaries of slaughtered crossbred beef heifers (under 30 months, n = 1241) and cows (over 4 years old, n = 1125), and processed in vitro as above. No significant difference was observed between the two groups in terms of the number of aspirated follicles or oocytes recovered. A significantly higher proportion (P < 0.01) of cow oocytes than heifer oocytes reached the blastocyst stage (Day 8: 46.5% versus 33.4%). In Experiment 3, ovaries were separated according to age of heifer into three groups: (1) 12-18 months, (2) 19-24 months and (3) 25-30 months, and compared with cow oocytes. There was no significant difference in the blastocyst yield between the different age groups of heifers. Irrespective of heifer age, the blastocyst yield on Day 8 was significantly lower than that from cow oocytes (35.0, 35.2, 36.5 and 48.3%, respectively, P < 0.05). In Experiment 4, a significantly higher proportion (P < 0.001) of presumptive zygotes derived from abattoir-derived cow oocytes reached the blastocyst stage following culture in vivo in the ewe oviduct than those derived from heifer oocytes (Day 8: 53.1% versus 25.2% for cow and heifer oocytes, respectively). In conclusion, the origin of the oocyte has a significant impact on its subsequent developmental potential. These results would suggest that in an in vitro production system, cow oocytes should be preferentially used over those from heifers in order to maximize blastocyst development.     

Developmental competence of oocytes from prepubertal Bos indicus crossbred cattle

The aim of the present study was to evaluate the effect of age on embryogenic competence of oocytes recovered from Bos indicus crossbred calves and heifers. Cumulus-oocyte complexes (COCs) were collected from 4- to 7-month-old calves (experiment 1) and from 9- to 14-month-old heifers (experiment 2) during processing at an abattoir. In both experiments cow COCs were used as control. COCs were in vitro matured and fertilized, and the presumptive zygotes co-cultured with cumulus cells until 224 h post insemination (hpi). In experiment 1, the development rate during the first 68-72 hpi was similar (P > 0.05) between embryos derived from calves and cows. Fewer embryos from calves developed to the blastocyst stage, resulting in a lesser blastocyst production as well as lesser hatching rate (P < 0.05). The embryo development after blastocyst stage was, nevertheless, similar (P > 0.05) between blastocysts derived from calves and cows, suggesting that the development after blastocoele formation is not compromised in embryos derived from calves. In experiment 2, there were no differences (P > 0.05) on cleavage, blastocyst and hatching rates between embryos derived from prepubertal heifers and cows. The rate of blastocyst development until hatching was also similar (P > 0.05). These results indicate that oocytes from 9- to 14-month-old B. indicus crossbred heifers have the same developmental competence as oocytes derived from cows, while ocytes derived from 4- to 7-month-old B. indicus crossbred calves are less competent in developing to the blastocyst stage in vitro. It suggests that oocyte competence in B. indicus crossbred cattle is achieved around 9-14 months of age.     

Effects of increasing diet fermentability on intake,digestion, rumen fermentation,blood metabolites and milk production of heat-stressed dairy cows

Heat stress is a major problem for dairy cows in hot climates, thus coping strategies are essential. This study evaluated the effects of increasing diet fermentability on intake, total tract digestibility, ruminal pH and volatile fatty acids (VFA) profile, blood metabolite profile and milk production and composition of lactating dairy cows managed under conditions of ambient heat stress. Nine multiparous cows (650 ± 56 kg BW; mean ± SD) averaging 102 ± 13 days in milk and producing 54 ± 6 kg/day were randomly assigned to a triplicate 3 × 3 Latin square. During each 21-day period, cows were offered one of three total mixed rations that varied in diet fermentability. The three levels of diet fermentability were achieved by increasing the proportion of pellets containing ground wheat and barley in the dietary DM from 11.7% (low), to 23.3% (moderate), and 35.0% (high) by replacing ground corn grain. Each period had 14 day of adaptation and 7 day of sampling. The ambient temperature–humidity index ( ≥ 72) indicated that the cows were in heat stress almost the entire duration of the study. Also, rectal temperature of cows was elevated at 39.2°C, another indication of heat stress. Increasing diet fermentability linearly decreased dry matter intake (22.8, 22.5, 21.8 kg/day for low, moderate and high, respectively; P ≤ 0.05) but increased non-fibre carbohydrate digestibility (P ≤ 0.05) and tended to increase digestibility of DM (P = 0.10) and crude protein (P = 0.06). As a result, the intake of digestible DM was not affected by the treatments. The production of 3.5% fat corrected milk (32.6, 33.7, and 31.5 kg/day) was quadratically (P ≤ 0.05) affected by diet fermentability with lower production for the high diet compared with the other two, which were similar. Rumen pH (ruminocentesis) and proportions of butyrate and isovalerate linearly decreased whereas propionate proportion linearly increased with increasing diet fermentability (P ≤ 0.05). The rumen concentration of NH₃-N (11.0, 9.0, and 8.7 mg/dL) and blood concentration of urea linearly decreased with increasing diet fermentability (P ≤ 0.05). The activity of alkaline phosphatase increased (65.1, 83.2, and 84.9 U/l) and concentration of malondialdehyde decreased (2.39, 1.90 and 1.87 µmol/l) linearly with increasing diet fermentability (P ≤ 0.05), which indicated possible attenuation of the effects of oxidative stress with increasing diet fermentability. Overall, a modest increase of diet fermentability improved nitrogen metabolism, milk protein production and oxidative stress of heat-stressed dairy cows, but a further increase in diet fermentability decreased milk yield.     

Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation

We investigated the effect of meiotic stages and two maturation protocols on bovine oocyte's resistance to cryopreservation. Oocytes at germinal vesicle breakdown (GVBD) and metaphase II (MII) stage as well as oocytes matured for 22 h in media supplemented with FSH or LH were vitrified by the open pulled straw method. After warming, oocytes underwent additional 16 h (GVBD group) or 2 h (MII group) maturation. Then they were subjected to in vitro fertilization and culture. Some oocytes that matured in the medium supplemented with LH were subjected to parthenogenetic activation after vitrification to determine their developmental potential in absence of fertilization. Survival of oocytes after vitrifying/warming was determined after 22 h in fertilization medium. Cleavage and blastocyst formation rates were used to assess their developmental competence. In both experiments, a portion of unvitrified MII oocytes were subjected to in vitro fertilization and culture as control groups. In Experiment 1, similar cleavage rates were obtained for both GVBD and MII oocytes (53.56 versus 58.01%, P > 0.05). However, significantly higher proportion of cleaved embryos from vitrified MII oocytes developed into blastocysts than those from vitrified GVBD oocytes (1.06 versus 8.37%, respectively, P < 0.01). In Experiment 2, vitrified MII oocytes matured in medium supplemented with LH were superior to vitrified MII oocytes matured in FSH supplementation not only in cleavage rates (61.13 versus 50.33%), but in blastocyst formation rates (11.79 versus 5.19%, P < 0.01) as well. Cleavage and blastocyst formation rates of parthenogenetically activated oocytes were similar to those that were fertilized. Nevertheless, the vitrifying/ warming process significantly compromised the oocytes' developmental capacity since the vitrified oocytes showed significant reduction in both cleavage and blastocyst rates compared to those of not vitrified controls in both experiments (P < 0.01). We showed that oocytes at different maturation stages respond to cryopreservation differently and MII stage oocytes have better resistance to cryopreservation than GVBD stage oocytes. The maturation protocols also influence oocyte's ability to survive cryopreservation. Poor developmental potential after vitrification seem to have resulted from the cryodamage to the oocyte itself. These results suggested the importance of maturation on the developmental competence of cryopreserved oocytes.     

Herbage intake and behavioural adaptation of grazing dairy cows by restricting time at pasture under two feeding regimes

The time at pasture of dairy cows is often restricted in the context of extending the grazing season in autumn or at the end of winter. The objective of our study was to evaluate the effects of a restriction of time at pasture on milk production, herbage intake and feeding behaviour in dairy cows according to feeding regime. The four treatments consisted of 4 h or 8 h of time at pasture per day tested under two feeding regimes combining rate of supplementation and herbage allowance: either a high rate of supplementation (10 kg dry matter (DM) of a maize silage-soya bean meal mixture in the ratio 87 : 13 on a % DM basis) with a low herbage allowance (6 kg DM/cow per day above 5 cm), or a low rate of supplementation (5 kg DM of the same supplement) with a high herbage allowance (11 kg DM/cow per day). The study was carried out according to a 4 × 4 Latin square design with four 2-week periods, with 48 mid-lactation Holstein cows. The cows in the 4-h treatment had access to pasture from 0900 h to 1300 h and those in the 8-h treatment from 0900 h to 1700 h. The supplement was given at 1830 h. When time at pasture was reduced from 8 h to 4 h per day, herbage intake decreased (9.9 v. 8.1 kg DM, P < 0.001), along with a fall in milk production (22.3 v. 21.2 kg, P < 0.001) and milk protein concentration (30.1 v. 29.6 g/kg, P < 0.001), while milk fat concentration increased (39.4 v. 39.9 g/kg, P < 0.05). The effect of time at pasture on milk production was slightly more marked on the low-supplement feeding regime (interaction P < 0.06). Reducing time at pasture by 4 h led to a sharp decrease in grazing time (327 v. 209 min, P < 0.001), but strongly increased the pasture intake rate (31 v. 39 g DM/min, P < 0.001) and the proportion of time spent grazing (0.68 v. 0.87, P < 0.001). Cows showed a stronger motivation for grazing when receiving the low-supplement feeding regime. In conclusion, we showed that reducing time at pasture from 8 to 4 h for cows receiving 5 to 10 kg DM of a maize silage-based supplement decreased moderately milk production and herbage intake, because of the capacity for behavioural adaptation by the grazing dairy cows.     

Apolipoprotein A-I, A-II, and VLDL-B-100 metabolism in men: comparison of a low-fat diet and a high-monounsaturated fatty acid diet

The impact of a low-fat diet and a high-MUFA diet on apolipoprotein A-I (apoA-I), apoA-II, and VLDL-apoB-100 metabolism in conditions of unrestricted (ad libitum) energy intake was compared in 65 men randomly assigned to one of two predefined experimental diets. A subsample of 18 men participated in the kinetic study. Before and after the 6-7 week dietary intervention, kinetic subjects received a primed-constant infusion of [5,5,5-2H3]L-leucine for 12 h under feeding conditions. ApoA-I production rate (PR; -31.5%; P <0.001) and fractional catabolic rate (FCR; -24.3%; P <0.05) were significantly decreased after the low-fat diet. These changes in apoA-I PR and FCR with the low-fat diet were also significantly different from those observed with the high-MUFA diet (P <0.01 and P <0.05, respectively). ApoA-II FCR was significantly increased in the high-MUFA group only. No significant within- or between-diet difference was found in VLDL-apoB-100 PR or FCR. These results emphasize the differential impact of the low-fat diet and high-MUFA diet on HDL metabolism.     

Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum

Recently, piglets have been obtained from in vitro-produced blastocysts by using in vitro maturation systems in which oocytes have been matured in North Carolina State University (NCSU) solution supplemented with porcine follicular fluid (PFF). However, PFF is not available commercially. To prepare PFF from the ovaries required time and effort and there is substantial variation in quality among batches. Furthermore, PFF is considered a potential source of infectious agents. We evaluated another commercially available potential protein source, fetal bovine serum (FBS), for in vitro maturation, to produce embryos and piglets. Cumulus-oocyte complexes were matured in NCSU-37 with PFF or with one of four batches of FBS. The proportions of oocytes with expanded cumulus cells were lower in all FBS batch groups (P < 0.05, 15-41%) than that in the PFF group (74%). The proportions of oocytes that matured were also lower in all FBS batch groups (P < 0.05, 26-41%) than in the PFF group (73%), irrespective of cumulus expansion. However, the proportions of oocytes that underwent germinal vesicle breakdown were almost the same in all groups (76-96%). After in vitro fertilization, the rate of sperm penetration into matured oocytes was higher in the PFF group (P < 0.05, 63%) than in one batch of FBS (22%) and removal of the compacted cumulus cells after maturation did not affect fertilization status (21%). Subsequent in vitro embryo culture of the PFF and FBS groups for 6 day resulted in similar rates of blastocyst formation (17 and 19%, respectively) and similar numbers of cells per blastocyst (43 and 46 cells, respectively). When blastocysts obtained from oocytes matured with FBS were transferred into two recipients, one became pregnant and farrowed seven piglets. Transfer of blastocysts obtained from oocytes matured with PFF into two other recipients resulted in one pregnancy and production of four piglets. These data suggested that porcine in vitro maturation in NCSU-37 supplemented with FBS reduced the maturational ability of oocytes, but once oocytes have matured, they have the same ability to develop to term after in vitro fertilization and embryo transfer as those matured with PFF.     

Relationship of morphology and follicular fluid environment of bovine oocytes to their developmental potential in vitro

Morphology of bovine cumulus oocyte complexes (BCOC) and follicular fluid parameters were examined as potential criteria for selecting in vitro developmentally competent oocytes. Immature BCOC, from antral follicles, exhibiting similar morphological characteristics were grouped and the blastocyst development for oocytes in each group was examined. In a second experiment, follicles were individually aspirated to recover BCOC with their associated follicular fluid. Each oocyte was categorized and individually matured, fertilized and cultured. Radioimmunoassays for follicular progesterone, estradiol-17beta and insulin-like growth factor-I (IGF-1) concentrations were performed. The BCOC were categorized into 9 groups based on the homogeneity of the ooplasm, compactness of the cumulus investment, color and size. Oocytes classified into Groups 1, 2 and 3 demonstrated the highest rates of development to the blastocyst stage at 13, 16 and 20%, respectively. Therefore, Groups 1, 2 and 3 were pooled and designated as the enhanced developmental potential (EDP) group, and the remaining groups were designated as the reduced developmental potential (RDP) group. The progesterone concentration (+/- SEM) in follicles containing EDP oocytes (27.54 +/- 5.08 ng/ml) was significantly lower than the RDP group (72.72 +/- ng/ml; P < 0.001). Follicles containing oocytes that developed to the blastocyst stage (28.65 +/- 7.18 ng/ml) possessed significantly lower progesterone levels than all remaining follicles (52.9 +/- 7.51 ng/ml; P < 0.05). No significant differences were found for estradiol-17beta or IGF-I concentrations between the EDP and RDP groups or developmental stages. These results indicate that morphological criteria and follicular fluid progesterone concentration may be used to select BCOC for high potential of blastocyst development.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Bovine immature oocytes acquire developmental competence during meiotic arrest in vitro

To test the hypothesis that oocytes require time to acquire developmental competence during meiotic arrest, we investigated the effects of butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinase, on the developmental competence of bovine oocytes after in vitro fertilization (IVF) following release from meiotic arrest. In the present study, 4 culture conditions were used: addition of BSA or fetal bovine serum (FBS) under 2 oxygen tensions (5% vs. 20%) during meiotic arrest with 100-microM BL I. The developmental competence to the blastocyst stage was higher (P < 0.01) in oocytes that were arrested in FBS-supplemented medium under 5% O2 (37%) than in oocytes that were arrested under other conditions (5%-24%) or that matured directly following follicle aspiration (23%). The time course of nuclear maturation of BL I-treated oocytes was also examined. The results demonstrated that oocytes treated with BL I start germinal vesicle (GV) breakdown and reach the metaphase II stage 5.5-6.0 h earlier than nonarrested oocytes. The developmental rates to the blastocyst stage of BL I-treated oocytes matured for 15.5 and 21 h were higher (P < 0.05) than those of nontreated oocytes matured for 21 and 26.5 h, respectively. These results demonstrate that bovine immature oocytes, which were arrested at the GV stage with BL I in FBS-supplemented medium under low oxygen tension, acquire higher developmental competence during meiotic arrest.     

Effects of Isoflavone-Enriched Feed on the Rumen Microbiota in Dairy Cows

In this study, we compared the effects of two diets containing different isoflavone concentrations on the isoflavone transfer from feed into milk and on the rumen microbiota in lactating dairy cows. The on-farm experiment was conducted on twelve lactating Czech Fleckvieh x Holstein cows divided into two groups, each with similar mean milk yield. Twice daily, cows were individually fed a diet based on maize silage, meadow hay and supplemental mixture. Control group (CTRL) received the basal diet while the experimental group (EXP) received the basal diet supplemented with 40% soybean isoflavone extract. The average daily isoflavone intake in the EXP group (16 g/day) was twice as high as that in the CTRL group (8.4 g/day, P<0.001). Total isoflavone concentrations in milk from the CTRL and EXP groups were 96.89 and 276.07 μg/L, respectively (P<0.001). Equol concentrations in milk increased from 77.78 μg/L in the CTRL group to 186.30 μg/L in the EXP group (P<0.001). The V3-4 region of bacterial 16S rRNA genes was used for metagenomic analysis of the rumen microbiome. The experimental cows exhibited fewer OTUs at a distance level of 0.03 compared to control cows (P<0.05) and reduced microbial richness compared to control cows based on the calculated Inverse Simpson and Shannon indices. Non-metric multidimensional scaling analysis showed that the major contributor to separation between the experimental and control groups were changes in the representation of bacteria belonging to the phyla Bacteroidetes, Proteobacteria, Firmicutes, and Planctomycetes. Surprisingly, a statistically significant positive correlation was found only between isoflavones and the phyla Burkholderiales (r = 0.65, P<0.05) and unclassified Betaproteobacteria (r = 0.58, P<0.05). Previous mouse and human studies of isoflavone effects on the composition of gastrointestinal microbial populations generally report similar findings.     

In vitro development of domestic cat embryos following intra-cytoplasmic sperm injection with testicular spermatozoa

The objective was to assess the ability of testicular spermatozoa to fertilize in vitro matured domestic cat oocytes and support blastocyst formation in vitro following intra-cytoplasmic sperm injection (ICSI). After IVM, oocytes were randomly and equally allocated among treatment groups (ICSI with testicular spermatozoa, ICSI with ejaculated spermatozoa, sham ICSI, and control IVF). At 18 h after either injection or insemination, the percentage of fertilized oocytes (per total metaphase II oocytes) was approximately 65% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which was less (P < 0.05) than control IVF (approximately 90%). On Day 7, the percentage of cleaved embryos (per total metaphase II oocytes) was approximately 60% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which also was less (P < 0.05) than control IVF (approximately 85%). After ICSI with testicular spermatozoa, the percentage of blastocysts (per total cleaved embryos) was approximately 11.0%, which was less (P < 0.05) than ICSI with ejaculated spermatozoa (approximately 21.0%); the latter was less (P < 0.05) than control IVF (approximately 43.0%). No blastocyst formation was observed after sham ICSI. For the first time in the domestic cat, this study demonstrated the fertilizing ability and developmental potential of intra-testicular spermatozoa delivered directly into intra-ovarian oocytes matured in vitro.     

Effect of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos

In order to develop a culture system and recipient cytoplasm that could improve the developmental competence of somatic cell nuclear transfer (SCNT) embryos for successful cloning of pigs, we evaluated the effect of donor oocytes and in vitro maturation (IVM) media on maturation of oocytes and developmental competence of SCNT embryos. In Experiment 1, oocytes derived from sows or gilts were matured in two IVM media (TCM-199 versus NCSU-23) and maturation of oocytes was evaluated by the status of chromatin configuration, the diameter of matured oocytes, the thickness of the zona pellucida, and the size of the perivitelline space (PVS). Sow oocytes matured in TCM-199 (S-TCM group) and NCSU-23 (S-NCSU group) showed significantly higher (P<0.05) maturation rates (S-TCM and S-NSCU, 86+/-4 and 82+/-4%, respectively) when evaluated by metaphase-II status than the gilt oocytes matured in TCM-199 (G-TCM group, 71+/-3%) and in NCSU-23 (G-NCSU-23 group, 71+/-3%). Oocyte diameter, the thickness of the zona pellucida, and the perivitelline space of sow oocytes (S-TCM and S-NCSU) were larger than those of gilt oocytes (G-TCM and G-NCSU) after IVM (P<0.05). In Experiment 2, SCNT was performed, using in vitro-matured oocytes from each group as recipient cytoplasm and porcine fetal fibroblasts as karyoplasts. The reconstructed embryos were electrically fused and activated, and cleavage and blastocyst formation were monitored under a stereomicroscope. The total cell number of flattened blastocysts stained with 5 microM bisbenzimide on day 7 were counted. In addition, in vitro matured non-enucleated oocytes were also electrically activated (parthenogenetic activation) and pronuclear formation was monitored. No difference in pronuclear formation rate after parthenogenetic activation and fusion rate after SCNT was observed among experimental groups. A significantly higher cleavage rate (P<0.05) was observed in S-TCM (69+/-4%) when compared with only G-NCSU (58+/-4%), but not with G-TCM (60+/-4%) or S-NCSU (68+/-4%). The rate of blastocyst formation was significantly higher (P<0.05) in sow oocytes (24% in S-TCM and S-NCSU), when compared to that observed in G-TCM (15%), and G-NCSU (14%). When the same source of oocytes was used, there was no significant difference in rate of blastocyst formation in the two culture media. Total cell number of blastocysts were not significantly different among experimental groups. In conclusion, the present study clearly demonstrated that sow oocytes have a greater developmental competence than gilt oocytes, regardless of the maturation medium examined.     

Cumulus cells during in vitro fertilization and oocyte vitrification in sheep: Remove,maintain or add?

The objective was to evaluate the developmental competence of immature and matured ovine oocytes after removing, maintaining or adding cumulus cells (CC) associated to vitrification by Cryotop method. Three experiments were performed involving 3,144 oocytes. In Experiment 1, CC were removed from immature, matured or fertilized oocytes subjected to in vitro embryo production. In Experiment 2, oocytes were vitrified either in MI or MII stage with or without CC, while a control group with CC remained unvitrified. In Experiment 3, oocytes partially denuded from CC were vitrified either in MI or MII stage, and a co-culture of fresh CC was added or not soon after warming to complete in vitro maturation (IVM) and in vitro fertilization (IVF), or IVF, respectively, while a control group remained unvitrified. In Experiment 1, the cleavage rate, development rate on Day 6 and blastocyst rate on Day 8 were improved when CC were maintained until the end of IVF (P < 0.05). In Experiment 2, vitrification of oocytes with enclosed CC showed a tendency to increase cleavage (P = 0.06) and improved blastocyst rate (P < 0.05). In Experiment 3, adding CC as co-culture after vitrification-warming tended to improve cleavage rate (P = 0.06) and increased hatching rate (P < 0.05). Regarding oocyte stage, vitrification of in vitro matured oocytes resulted in greater developmental competence than immature stages (P < 0.05). In conclusion, CC seems to have a relevant role for in vitro embryo development in either fresh or vitrified oocytes.     

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology,fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.     

Low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine cumulus-oocyte complexes

The effects of carbohydrates on meiotic maturation and ATP content of bovine oocytes under low oxygen tension (5%) were investigated. Furthermore, the developmental competence or intracellular H(2)O(2) contents of the oocytes matured under 5% or 20% O(2) was assessed. In vitro maturation of bovine cumulus-oocyte complexes was performed in synthetic oviduct fluid (SOF) containing 20 amino acids and hormones (SOFaa). The proportion of the oocytes that matured to the metaphase II stage in SOFaa containing 1.5 mM glucose, 0.33 mM pyruvate, and 3.3 mM lactate under 5% O(2) was dramatically lower than that of oocytes matured under 20% O(2) (P < 0.01). Similarly, the ATP content of the oocytes that matured under 5% O(2) was much lower than that of oocytes matured under 20% O(2) (P < 0.05). Under 5% O(2) the proportion of metaphase II oocytes increased with increasing glucose concentration (0-20 mM) in SOFaa without pyruvate or lactate. In addition, the ATP content of oocytes cultured in 20 mM glucose was higher (P < 0.05) than that of oocytes cultured in 1. 5 mM glucose. Two glucose metabolites (pyruvate and lactate) and a nonmetabolizable glucose analog (2-deoxy-glucose), however, had no noticeable effects on meiotic maturation under 5% O(2). These results suggest that ATP production under 5% O(2) is not dependent on the TCA cycle. Addition of iodoacetate, a glycolytic inhibitor, to SOFaa containing 20 mM glucose significantly reduced (P < 0.01) the proportion of metaphase II and ATP content. Moreover, the proportion of the development to the blastocyst stage of oocytes matured under 5% O(2) was higher (P < 0.05) than that of oocytes matured under 20% O(2). H(2)O(2) contents of oocytes matured under 5% O(2) was lower (P < 0.05) than that of oocytes matured under 20% O(2). The results of the present study demonstrate that glucose plays important roles in supporting the completion of meiotic maturation in bovine cumulus-oocyte complexes under low oxygen tension and that low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine oocytes.     

A high-fat diet raises fasting plasma CCK but does not affect upper gut motility, PYY, and ghrelin, or energy intake during CCK-8 infusion in lean men

There is evidence from studies in animals that the effects of both fat and CCK on gastrointestinal function and energy intake are attenuated by consumption of a high-fat diet. In humans, the effects of exogenous CCK-8 on antropyloroduodenal motility, plasma CCK, peptide YY (PYY), and ghrelin concentrations, appetite, and energy intake are attenuated by a high-fat diet. Ten healthy lean males consumed isocaloric diets (~15,400 kJ per day), containing either 44% (high-fat, HF) or 9% (low-fat, LF) fat, for 21 days in single-blind, randomized, cross-over fashion. Immediately following each diet (i.e., on day 22), subjects received a 45-min intravenous infusion of CCK-8 (2 ng.kg(-1).min(-1)), and effects on antropyloroduodenal motility, plasma CCK, PYY, ghrelin concentrations, hunger, and fullness were determined. Thirty minutes after commencement of the infusion, subjects were offered a buffet-style meal, from which energy intake (in kilojoules) was quantified. Body weight was unaffected by the diets. Fasting CCK (P < 0.05), but not PYY and ghrelin, concentrations were greater following the HF, compared with the LF, diet. Infusion of CCK-8 stimulated pyloric pressures (P < 0.01) and suppressed antral and duodenal pressures (P < 0.05), with no difference between the diets. Energy intake also did not differ between the diets. Short-term consumption of a HF diet increases fasting plasma CCK concentrations but does not affect upper gut motility, PYY and ghrelin, or energy intake during CCK-8 infusion, in a dose of 2 ng.kg(-1).min(-1), in healthy males.     

Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice

Exposure to high-fat diets for prolonged periods results in positive energy balance and obesity, but little is known about the initial physiological and neuroendocrine response of obesity-susceptible strains to high-fat feeding. To assess responses of C57BL/6J mice to high- and low-fat diets, we quantitated the hypothalamic expression of neuropeptides implicated in weight regulation and neuroendocrine function over a 2-wk period. Exposure to high-fat diet increased food consumption over a 2-day period during which leptin levels were increased when assessed by a frequent sampling protocol [area under the curve (AUC): 134.6 +/- 10.3 vs. 100 +/- 12.3, P = 0.03 during first day and 126.5 +/- 8.2 vs. 100 +/- 5.2, P = 0.02 during second day]. During this period, hypothalamic expression of neuropeptide Y (NPY) and agouti-related protein (AgRP) decreased by approximately 30 and 50%, respectively (P < 0.001). After 1 wk, both caloric intake and hypothalamic expression of NPY and AgRP returned toward baseline. After 2 wk, cumulative caloric intake was again higher in the high-fat group, and now proopiomelanocortin (POMC) was elevated by 76% (P = 0.01). This study demonstrates that high-fat feeding induces hyperphagia, hyperleptinemia, and transient suppression of orexigenic neuropeptides during the first 2 days of diet. The subsequent induction of POMC may be a second defense against obesity. Attempts to understand the hypothalamic response to high-fat feeding must examine the changes as they develop over time.