Liver Condition Affects Bovine Oocyte Qualities by Changing the Characteristics of Follicular Fluid and Plasma

The liver is an important organ that contributes to milk production in dairy cows. The aim of this study was to examine whether liver conditions affect the characteristics of blood plasma and follicular fluid (FF) and whether supplementing in vitro maturation medium with FF from either cows with damaged livers (DL) or those with healthy livers (HL) affects oocyte developmental competence. Biochemical characteristics of FF were significantly correlated with those in plasma. As such, the characteristics of both plasma and FF were similarly affected by liver conditions in that the concentrations of total protein and inorganic phosphorus were higher for the DL cow group than for the HL cow group, whereas the concentrations of albumin, lactate dehydrogenase and calcium were lower for DL cows than for HL cows. In addition, supplementing the medium with DL‐FF retarded the progression of the nuclear maturation of oocytes collected from the HL cows. On culturing oocytes in maturation medium containing HL‐FF, DL‐FF or foetal calf serum, the highest developmental rate to the blastocyst stage was observed in the HL‐FF group, while the lowest developmental ratio was observed in the DL‐FF group. The growth factor array of the FFs revealed that 10 growth factors were significantly downregulated in the DL‐FF compared with those in HL‐FF. In conclusion, the characteristics of plasma and FF are affected by liver conditions in a similar way. Concentrations of several growth factors were low in DL‐FF, as was the ability of DL‐FF to support oocyte maturation compared with that of HL‐FF.     

Acid–Base Parameters and Steroid Concentrations in Pre‐Ovulatory Follicles and Plasma of Lactating Dairy Cows with Spontaneous and Synchronized Oestrus or Follicular Cyst

The study aimed to compare the acid–base balance and steroid concentrations between follicular fluids (FF) of pre‐ovulatory follicles derived from a spontaneous oestrus (SO), synchronized or induced oestrus (IO) and follicular cysts (CYS) and between FF and blood in dairy cows. Forty‐two dairy cows were included in this study. The animals were allocated to three groups: SO (n = 23); IO (n = 11) using GnRH at day 0 and day 9 and PGF₂α at day 7; and animals with CYS (n = 10). The follicular fluids (FF) were aspirated from the cyst/pre‐ovulatory follicles (? ≥ 15 mm) after SO and after second GnRH dose in IO by transvaginal ultrasound‐guided ovum pick‐up technique. Blood samples (BL) were collected in heparinized vacutainer tubes. The oxygen tension (pO2) in FF of IO was higher (p < 0.05) than in SO and CYS groups. There were negative correlations (p < 0.001, r = ?0.89) between FF and blood pO2. The carbon dioxide tension (pCO2) and lactate level in FF of CYS group were higher (p < 0.05) than in SO and IO groups. There were negative correlations (p < 0.01, r = ?0.73) between blood and FF pO2. Oestradiol‐17β concentration in pre‐ovulatory follicles and plasma of the SO group was higher (p < 0.001) than in IO and CYS groups. Progesterone concentration in pre‐ovulatory follicles and plasma of the SO and IO groups was lower (p < 0.01) than in CYS group. Plasma androstenedione concentration in SO and IO groups was higher (p < 0.05) than in CYS group. In conclusion, acid–base parameters, E2 and P4 levels in the follicular fluid of both IO and CYS groups were deviated greatly from the physiological level (disturbances of intrafollicular/intracystic environment), which may affect the quality of both the oocyte and the granulosa cells.     

In vitro Development of Buffalo Oocytes in Media-containing Fluids from Different Size Class Follicles

Studies were conducted to investigate the effect of supplementation of fluid from different sized class [small (SFF, < 3 mm), medium (MFF, 3-8 mm) and large (LFF, > 8 mm)] of normal and cystic (CFF) ovarian follicles in oocyte culture media on oocyte maturation rate and embryo development in vitro and to test the efficacy of follicular fluid (FF) from different size classes as a whole oocyte maturation medium. Results suggested that FF were capable of developing buffalo oocytes to embryonic stage in vitro although its efficacy was lower than that of serum. Regardless of high maturation rates after in vitro maturation (IVM) in media containing FF or IVM in whole FF, low blastocyst rates were obtained after in vitro fertilization (IVF) and culture of embryos. Follicular fluid from small follicles had significantly (p < 0.05) higher potential of developing buffalo oocytes to embryonic stage in vitro than that from medium and large follicles. Cystic FF was not capable of supporting development of buffalo oocytes in vitro.     

Anomalous Pregnancies during Late Embryonic/Early Foetal Period in High Producing Dairy Cows

This study analyses anomalous cases of gestation ending in pregnancy loss during the early foetal period and their effect on progesterone and plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations. Data derived from a large-scale ultrasound pregnancy diagnosis programme in high producing dairy cows. Over a 3-year period (2004–2007), a very low incidence (0.5%: 15 of 3094) of anomalous pregnancies was recorded. The results revealed that the following anomalies were detected on days 35–41 of gestation in cows carrying singletons with one single corpus luteum: embryo death in eight cows (0.3%); and embryo in the uterine horn contralateral to the corpus luteum in seven cows (0.2%). All these animals suffered pregnancy loss during the early foetal period. In cows carrying dead embryos, no signs of conceptus degeneration were observed on pregnancy diagnosis. Amnion size (approximately 25 mm diameter) and uterine horn fluid contents were estimated to be similar to those of the normal pregnant cows in this period. In the contralateral gestations, live embryos were observed in all ultrasound checks before pregnancy loss. Uterine fluid contents increased in the two cows in which gestation continued for more than a week. In the cases of embryo death but not in those of contralateral gestation, a drop in PAG-1 levels was noted prior to pregnancy loss. Two cows carrying dead embryos increased with time allantoic fluid contents. The PAG-1 values increased with time in one cow bearing a dead embryo (from 2.31 to 6.79 ng/ml) and in two of the contralateral gestations (from 1.66 to 2.33 ng/ml and from 0.39 to 6.79 ng/ml, respectively). Results of this study indicate that the foetal membranes continue to undergo some activity following embryo death, and that contralateral pregnancy may determine failure of the gestation process.     

Relationship between oxidative stress and the success of artificial insemination in dairy cows in a pasture-based system

This study was designed to evaluate whether the outcome of artificial insemination (AI) was affected by the metabolic and oxidative status of dairy cows. Seventy-nine inseminations in 40 cows, were classified, on the basis of blood progesterone (P4) and pregnancy-associated glycoprotein (PAG) concentrations and clinical confirmation of pregnancy into, three categories: (1) positive (AI+, resulted in pregnancy, n=26; 33%), (2) negative (AI-, did not result in pregnancy, n=49; 62%), and (3) embryonic mortality (EM, n=4; 5%). Reactive oxygen metabolites, biological antioxidant potential, oxidative stress index, body condition score, glucose, total proteins, albumin, urea, non-esterified fatty acids (NEFAs), cholesterol, triglycerides, haptoglobin and advanced oxidative protein products (AOPPs) were measured on the day of AI (day 0), and 30 and 42days later. Cows with EM had lower BCS scores (2.5) than AI+ (2.8) and AI- (2.9) cows (P<0.05). During the post-partum period, body condition score (BCS) increased and NEFAs decreased (P<0.05) suggesting a recovery from the negative energy balance (NEB). The only significant differences found were that the mean concentration of AOPPs was higher and that of albumin lower in EM cows than in AI+ and AI- (P<0.05) animals. Plasma concentration of reactive oxygen metabolites and biological antioxidant potential were not related to AI outcome. Further studies are required to confirm this finding and to clarify the role of oxidative status on cows' fertility.     

Effects of charcoal-extracted follicular fluid on reproductive function in postpartum cows

In order to determine the role of follicle-stimulating hormone (FSH) on the resumption of ovarian function in cows early postpartum (PP), bovine follicular fluid (FF) was used to selectively suppress concentrations of FSH. Calves were removed from all cows within 24 hr of birth. Follicular fluid that was treated with charcoal to remove steroids (15 ml; n = 14) or serum (S) from an ovariectomized cow (15 ml, n = 14) was injected i.m. twice daily from days 1 to 10 PP. Blood samples were collected before each injection and frequent samples (every 15 min for 6 hr) taken on days 5 and 10 PP. Eight cows from each group (FF and S) were slaughtered on the morning of day 11 PP and pituitaries and ovaries collected. The remaining cows (n = 6) were observed for estrus. Treatment with FF delayed follicular growth (P less than 0.01), as evidenced by the largest follicle per cow observed at time of slaughter (3.6 +/- 0.42 vs 11.5 +/- 1.77 mm dia; FF vs S). The intervals from parturition to first estrus (P less than 0.11) and to first progesterone rise (25.3 +/- 1.97 vs 18.0 +/- 3.62 d; P less than 0.06) tended to be delayed by treatment with FF vs S. Many of the cows treated with S ovulated by day 10 PP, they were divided retrospectively into those that had ovulated by (n = 9) or after (n = 5) day 10 PP for analysis. Cows treated with FF had lower (P less than 0.05) and less variable (P less than 0.01) serum FSH concentrations while levels of luteinizing hormone (LH) tended (P less than 0.08) to be greater on days 5 and 10 PP. Follicular fluid decreased levels of FSH (P less than 0.001), but not LH (P less than 0.15), in the samples obtained twice daily compared to S-treated cows that did not ovulate by day 10 PP. Anterior pituitaries were dissociated, and cells from each cow were cultured in order to ascertain whether treatment with FF in vivo would affect gonadotropin secretion in vitro. Estradiol-17 beta (E) was incubated with pituitary cells to determine the effect of E on gonadotropin secretion from cells of PP cows, and to ascertain whether treatment with FF in vivo and with E in vitro would interact to affect secretion of FSH and LH in culture. After 2 d of incubation, cells were treated with 10(-9) M E or vehicle (1% ethanol).(ABSTRACT TRUNCATED AT 400 WORDS)     

The In Vitro Development of Bovine Oocytes after Maturation in Glucose and β-Hydroxybutyrate Concentrations Associated with Negative Energy Balance in Dairy Cows

Negative energy balance (NEB) in high yielding dairy cows early postpartum may affect oocyte quality. Therefore, we tested the effect of two different beta-hydroxybutyrate (BHB) and glucose concentrations, which are associated with subclinical or clinical ketosis, during in vitro maturation (IVM) on the developmental competence of bovine oocytes. In Expt 1, subclinical ketosis conditions were imitated. Oocytes were matured in four different serum-free media with two glucose concentrations (g1=2.75 mm or G1=5.5 mm glucose) and with or without BHB (BHB1=1.8 mm BHB). Following maturation groups were used: g1, G1, g1:BHB1 and G1:BHB1. In Expt 2, clinical ketosis conditions were mimicked by using the concentrations: g2=1.375 mm or G2=3.1 mm glucose and BHB2=4.0 mm BHB. The combinations used were: g2, G2, g2:BHB2 and G2:BHB2. After IVM and in vitro fertilization (IVF), presumptive zygotes were routinely cultured for 7 days in synthetic oviduct fluid [SOF; 5% fetal calf serum (FCS)]. At 48 h and 8 days pi, cleavage rate and number of blastocysts were recorded respectively. The results demonstrated that the maturation conditions mimicking subclinical (g1:BHB1) and clinical ketosis (g2 : BHB2) resulted in an impaired developmental competence of the oocyte after maturation. Especially the moderately low (g1) or extremely low glucose (g2) concentrations were responsible for this detrimental effect that was associated with a blocked cumulus expansion. Only in moderately low glucose conditions (g1:BHB1), BHB exerted an additive toxic effect during oocyte maturation resulting in a reduced blastocyst rate. Conclusively, our results may suggest that subclinical and clinical ketosis can affect the oocyte's developmental competence most likely through a directly adverse effect of the low glucose concentrations on oocyte maturation. Only in subclinical conditions this harmful effect may be aggravated by BHB.     

Influence of Body Condition on Serum Metabolic Indicators of Lipid Mobilization and Oxidative Stress in Dairy Cows During the Transition Period

The objectives of this study were to examine the influence of body condition of cows on metabolic and antioxidative status, as well as to investigate the relationship between metabolic indicators of lipid mobilization and oxidative stress during transition period. The study was conducted on 24 Holstein‐Friesian dairy cows divided into 2 groups according to their body condition score (BCS) as optimal (n = 12; BCS from 3.25 to 3.75) or adipose (n = 12; BCS ≥4). Metabolic status (glucose, triglycerides, total cholesterol, HDL cholesterol, NEFA and BHB), paraoxonase‐1 (PON1) and apolipoprotein A‐I (ApoA‐I) were analysed in sera taken on days ?30, ?10, ?2, 0, 5, 12, 19, 26 and 60 relative to parturition. Adipose cows had significantly higher glucose concentration at parturition being significantly decreased after parturition on days 12 and 19. Total cholesterol and HDL‐C concentrations were the lowest at parturition and significantly higher on days 26 and 60 after parturition in both groups of cows. Both investigated groups had significantly higher NEFA concentration from parturition until day 19 after parturition, indicating energy deficit and an increased lipid mobilization after calving. There were no significant differences in BHB concentration during transition period in both groups. No significant differences were found in PON1 activity and ApoA‐I concentration during transition period in both groups of cows. However, in adipose cows, although not significantly different, PON1 was decreased from calving until day 19 after parturition indicating a disturbance in antioxidative status in adipose cows. PON1 significantly positively correlated with total cholesterol and HDL‐C concentrations and negatively with NEFA indicating a strong relationship of PON1 with lipid metabolism. Significant positive correlation between NEFA and BHB in both groups of cows points out on energy deficit during transition period that cows tend to overcome by lipid mobilization providing alternative source of energy needed for parturition and lactation.     

A Field Study to Unravel Factors that are Significantly Associated with the Secretory Activity of the Corpus Luteum During the First Three Postpartum Cycles in High Yielding Dairy Cows,Based on the Amount of Steroidogenic and Endothelial Cells Present in the Luteal Tissue

Fourteen multi‐ and eight primiparous high‐yielding dairy cows were followed from the first till the fourth ovulation postpartum. Cows were randomly divided into two groups and supplemented with soybean (group I; n = 11) or rapeseed meal (group II; n = 11). Both groups were subjected to a biopsy sampling of the corpus luteum (CL) at cycle day 9. The luteal capillary network (visualized by Bandeiraea simplicifolia) was denser in cycles 2 and 3 (p = 0.0005). The same was seen for the surface occupied by steroidogenic cells (visualized by 3β‐hydroxysteroiddehydrogenase) (p = 0.0001). The peripheral blood progesterone concentration showed an increasing trend with increasing cycle number and was higher in primiparous cows (p = 0.013), which had also larger glands on cycle day 9. The area occupied by endothelial cells was positively correlated with the area occupied by steroidogenic cells (r = 0.59; p < 0.0001). Both the areas occupied by endothelial and by steroidogenic cells were negatively correlated with the blood concentration of nonesterified fatty acids (NEFAs) (respectively, r = ?0.377; p = 0.004 and r = ?0.355; p = 0.007). We can conclude that primiparous cows generally have higher peripheral progesterone levels during the first three cycles after calving which is associated with a larger CL. In comparison with those of the first post‐partum cycle, corpora lutea of cycles 2 and 3 have a denser capillary network and a larger area of steroidogenic cells, while these are only associated with a trend of higher peripheral progesterone concentrations.     

Impact of bovine somatotropin administration beginning at day 70 of lactation on serum metabolites, milk constituents, and production in cows previously exposed to exogenous somatotropin.

Metabolic and production responses are reported for 72 cows treated with bovine somatotropin (BST) for 30 days starting at day 70 of lactation. Of these 72 cows, 48 had been exposed in the preceding lactation to long-term treatment with BST at 3 dosages and 24 (controls) had not been given BST. Approximately half of the cows in each group were parity-2 cows, the rest were older. Comparisons between groups were made separately for parity-2, and older cows. Analyses, using pretreatment values of each variable as a covariate, indicated that older cows, but not parity-2 cows, significantly (P less than 0.05) increased milk production during treatment. Parity-2 cows, however, had a significantly higher milk fat percentage than controls following treatment. Cows treated with 51.6 or 86 mg BST/d in both parity groups had significantly higher serum-free fatty acids than controls. Estimated net energy balances were significantly lower for older treated cows, but did not significantly differ from controls for parity-2 treated cows. Older cows in the 86 mg of BST/d group tended to have higher concentrations of blood glucose than did older control-group cows. Treatment with BST did not significantly increase serum ketone concentrations in any group of animals, and none of the cows developed clinical ketosis during this period. Estimated net energy balance (ENEB) during treatment was a significant (P less than 0.05) covariate for free fatty acid concentrations in older cows and for milk fat percentage in parity-2 cows. Covariate adjusted analyses, using ENEB during treatment as a covariate, indicated that lipolytic stimuli already acting may be enhanced by treatment with BST, but a negative energy balance was not a necessary precondition for free fatty acid concentrations to increase following somatotropin treatment. Similarly, milk fat percentages for parity-2 treated cows were significantly (P less than 0.05) higher during treatment than controls when ENEB during treatment was used as a covariate. Increased milk fat concentrations in parity-2 treated cows were not associated with significant increases in the ratio of C18:C4-10 milk fatty acids, indicating that increased milk fat resulted from either an increase in incorporation of C18 fatty acids into milk fat coupled with an increase in de novo mammary synthesis of C4-10 milk fatty acids or an increase in C12-16 fatty acids that may arise either from increased tissue mobilization, from diet, or from de novo mammary synthesis.     

The efficiency of in vitro ovine embryo production using an undefined or a defined maturation medium is determined by the source of the oocyte

In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid.     

The effect of milk production level on host resistance of dairy cows, as assessed by the severity of experimental Escherichia coli mastitis

This study investigated the possible effects of milk production level on the host resistance of dairy cows. High (n = 18) and low (n = 18) producing cows on a research farm, which respectively produced 11 443 and 7 727 kg milk in their previous lactation, were compared. To enhance the possible differences in host resistance between high and low producing cows, the animals in both groups were metabolically stressed by overfeeding during the dry period or were fed according to requirements, resulting in four groups of nine cows. The metabolic status was monitored from two weeks pre-partum until 2.5-4.5 weeks post-partum. Host resistance was assessed by measuring the severity of experimentally induced Escherichia coli mastitis. Pre-partum blood glucose levels tended to be higher in overfed cows than in cows fed according to requirements. The post-partum energy balance was significantly more negative in high producing cows than in low producers, and tended to be more negative in overfed cows compared to cows fed according to the requirements. Post-partum plasma glucose, NEFA, beta-OH-butyrate and urea concentrations were similar in the four groups. Plasma glucose concentrations were significantly lower and liver triacylglycerol concentrations were significantly higher in third than in second parity cows. Host resistance was not affected by the production level or feeding regimen. There were no significant correlations between the metabolic status and the severity of experimental E. coli mastitis, except for the relatively more severe mastitis in the cows with beta-OH-butyrate concentrations above 1.4 mmol/L. In conclusion, milk production level did not affect host resistance in dairy cows, as measured by the severity of experimental E. coli mastitis. Even in a situation where cows were metabolically stressed by overfeeding, high producers were as able as low producers to cope with the demands of milk production, without consequences for host resistance.     

Consequences of Nitric Oxide Synthase Inhibition During Bovine Oocyte Maturation on Meiosis and Embryo Development

The importance of nitric oxide synthase (NOS) in bovine oocyte maturation was investigated. Oocytes were in vitro matured with the NOS inhibitor N^w- l -nitro-arginine methyl-ester (10⁻⁷, 10⁻⁵ and 10⁻³  m l -NAME) and metaphase II (MII) rates and embryo development and quality were assessed. The effect of l -NAME (10⁻⁷  m ) during pre-maturation and/or maturation on embryo development and quality was also assessed. l -NAME decreased MII rates (78–82%, p < 0.05) when compared with controls without l -NAME (96%). Cleavage (77–88%, p > 0.05), Day 7 blastocyst rates (34–42%, p > 0.05) and total cell numbers in blastocysts were similar for all groups (146–171 cells, p > 0.05). Day 8 blastocyst TUNEL positive cells (3–4 cells) increased with l -NAME treatment (p < 0.05). For oocytes cultured with l -NAME during pre-maturation and/or maturation, Day 8 blastocyst development (26–34%) and Day 9 hatching rates (15–22%) were similar (p > 0.05) to controls pre-matured and matured without NOS inhibition (33 and 18%, respectively), while total cell numbers (Day 9 hatched blastocysts) increased (264–324 cells, p < 0.05) when compared with the controls (191 cells). TUNEL positive cells increased when NOS was inhibited only during the maturation period (8 cells, p < 0.05) when compared with the other groups (3–4 cells). NO may be involved in meiosis progression to MII and its deficiency during maturation increases apoptosis in embryos produced in vitro . Nitric oxide synthase inhibition during pre-maturation and/or maturation affects embryo quality.     

Reduced Fertility in High‐yielding Dairy Cows: Are the Oocyte and Embryo in Danger? Part II Mechanisms Linking Nutrition and Reduced Oocyte and Embryo Quality in High‐yielding Dairy Cows*

Dairy cow fertility has been declining during since the mid‐80s and this has given rise to numerous scientific studies in which important parts of the pathogenesis are elucidated. Reduced oocyte and embryo quality are acknowledged as major factors in the widely described low conception rates and in the high prevalence of early embryonic mortality. Apart from the importance of the negative energy balance (NEB) and the associated endocrine and metabolic consequences, there is a growing attention towards the effect of the milk yield promoting diets which are rich in energy and protein. Starch‐rich diets can improve the energy status and thus the ovarian activity in the early postpartum period but the oocyte and embryo quality can suffer from such insulinogenic diets. Supplementation of dietary fat has a similar dual effect with a beneficial stimulation of the ovarian steroid production while the oocyte and the embryo display an altered energy metabolism and excessive lipid accumulation. High‐protein diets can elevate the ammonia and urea concentrations in the blood, leading to changed intrafollicular, oviductal and uterine environments. Oocytes and embryos are highly sensitive to such changes in their microenvironment, possibly leading to a disturbed maturation, fertilization or early cleavage. Several nutrition‐linked mechanisms, through which oocyte and/or embryo quality can be affected in modern dairy cows, well after the period of NEB, are proposed and comprehensively reviewed in the present report.     

Bovine OPU‐Derived Oocytes can be Matured In Vitro for 16–28 h with Similar Developmental Capacity

The aim of this study was to determine the optimal maturation culture period of ovum pick up (OPU)‐derived cumulus oocytes complexes (COCs) in relation to their developmental capacity. Embryo production, embryo cryotolerance, post‐transfer embryonic survival and calf characteristics such as gestation length, birthweight and sex ratio were investigated. This retrospective study covers the analyses of ovum pick up –in vitro production and calving results from a commercial programme that took place between March 1994 and September 2004. Donors were both heifers (of which approximately 90% pregnant) and cows (of which approximately 10% pregnant). Embryo production analyses were based on 7800 OPU sessions conducted from January 1995 until January 1999. Analyses of calving rate were based on 13 468 embryo transfers performed during January 1995 until May 2002. Analyses on calf characteristics were based on 2162 calves born between March 1994 and September 2004. The in vitro maturation culture period ranged from 16 to 28 h. The mean production rate of transferable embryos was 16.5% (1.2 embryos per OPU session). Length of maturation culture period did not affect the production of transferable embryos. Mean calving rate was 40.9% and 38.7% for fresh and frozen/thawed embryos, respectively. Calving rate was not affected by the maturation culture period. Mean birthweight, gestation length and proportion of male calves were 46 kg, 281.9 days and 52.8%, respectively. Maturation culture period did not affect these variables. In conclusion, this study shows that the in vitro maturation culture period within the range of 16–28 h does not affect in vitro embryo production, embryo cryotolerance, post‐transfer embryonic survival and calf characteristics, suggesting that all COC batches collected by OPU on the same day, can be fertilized in one IVF session without a significant loss in the production from oocyte to calf.     

Effect of Prepartum Energetic Supplementation on Productive and Reproductive Characteristics, and Metabolic and Hormonal Profiles in Dairy Cows under Grazing Conditions

The effect of cracked corn grain supplementation (3.5 kg/day) during 3 weeks before the expected calving date on milk production and composition, body condition score (BCS), metabolic and hormonal profiles and length of postpartum anoestrus was evaluated in multiparous Holstein dairy cows under grazing conditions (Energy supplemented group, n = 10; Control group, n = 10). Body condition score was weekly recorded during the peripartum period, from days −21 to +35 (parturition = day 0). Non-esterified fatty acids, β-hydroxybutyrate, cholesterol, urea, insulin, insulin-like growth factor I (IGF-I), leptin, thyroxine (T₄) and 3,3'5-triiodothyroinine (T₃) were weekly determined in plasma from days −21 to +35. The reinitiation of ovarian cyclicity was twice weekly determined by ovarian ultrasonography and confirmed by plasma progesterone concentrations. Cows fed energy concentrate prepartum had higher BCS during the prepartum and postpartum and produced more milk. Non-esterified fatty acids plasma concentrations were significantly higher in the energy group, while cholesterol was higher in the control group. Treated cows had higher levels of plasma insulin, IGF-I and leptin pre-calving. IGF-I, leptin and T₄ were diminished during the early postpartum period in both groups. Insulin levels were also diminished in the control group, but levels remained high in the energy-supplemented group. Treated cows ovulated sooner after parturition than controls. We conclude that Energetic supplementation prepartum in cows under grazing conditions increased milk production and reduced the reinitiation of ovarian activity, consistent with a better EB (BCS), higher prepartum levels of IGF-I, leptin and insulin, and higher insulin levels during early postpartum.     

Metabolic changes in cows with or without retained fetal membranes in transition period

The purpose of the study was to evaluate the effect of retained fetal membranes (RFM) on serum minerals and energy- and protein-related metabolites in dairy cows at a herd with a recent history of fatty liver syndrome. Forty-seven multiparous Holstein cows were selected during transition period. Nine cows had RFM longer than 24 h after calving. Blood samples were obtained on prepartum days 21 and 7 and postpartum days 7 and 21. We used repeated measure procedure of anova to evaluate the effect of RFM on serum metabolites. Cows with RFM had significantly higher concentrations of beta-hydroxybutyrate, non-esterified fatty acids and triglycerides after calving, but had lower concentrations of cholesterol during transition period. The concentrations of serum albumin and blood urea nitrogen were also significantly lower in RFM-affected cows than non-affected ones after parturition. Our results suggested that negative energy balance (NEB) postpartum was associated with RFM in dairy cattle. However, our findings did not reveal a cause and effect relationship with respect to the role of NEB as a possible risk factor for RFM.     

The Effect of Seasonal Thermal Stress on Lipid Mobilisation,Antioxidant Status and Reproductive Performance in Dairy Cows

Heat stress is a major factor contributing to low fertility of dairy cows with a great economic impact in dairy industry. Heat‐stressed dairy cows usually have reduced nutrient intake, resulting in a higher degree of negative energy balance (NEB). The aim of this study was to investigate the seasonal thermal effect on lipid metabolism, antioxidant activity and reproductive performance in dairy cows. Thirty‐two healthy dairy heifers were included in the study. According to the ambient temperature, animals were divided into two groups: winter (N = 14) and summer season (N = 18). Metabolic parameters, paraoxonase‐1 (PON1) activity and total antioxidant status (TAS) were monitored at the time of insemination (basal values) and from 1 week before until 8 weeks after calving. Number of services per conception and calving‐to‐conception (CC) interval were calculated from the farm recording data. Serum triglyceride, non‐esterified fatty acids (NEFA) and beta‐hydroxybutyrate (BHB) concentrations were significantly increased after calving in summer compared to winter, indicating higher degree of NEB in cows during summer. PON1 activity was significantly decreased after calving in both summer and winter group. TAS concentration was significantly lower in summer than that in winter. A significantly higher number of services were needed for conception in summer compared to winter, and CC interval was significantly longer in summer than that in winter as well. Additionally, reproductive performance significantly correlated with the severity of NEB, suggesting that lipid mobilization and lower antioxidant status contributed to poor reproduction ability in dairy cows during hot months.     

Effect of various dietary fat supplementations on liver lipid and glycogen of high-yielding dairy cows in the peripartal period

In a model experiment, Holstein-Friesian dairy cows were fed on a corn-silage-based diet supplemented with 11.75 MJ NE1 per day of calcium soaps of palm oil fatty acids (CAS) or hydrogenated triglyceride (HTG) or without fat supplementation (control). All diets were fed to the cows over a period from 21 +/- 3 days (d) prior to the expected calving to d 100 +/- 5 postpartum. On d 25 (basal sample) and d 14 prepartum as well as on d 5 and 25 postpartum liver samples were collected by percutaneous biopsy. Total lipid content, fatty acid composition and glycogen of liver tissues were determined. At d 5 postpartum, both control and CAS cows had higher liver lipid (P < 0.05) and lower glycogen (P < 0.05) concentrations than cows in the HTG group. No significant (P < 0.05) differences were detected in liver fat content among the groups at d 14 prepartum or d 25 postpartum. The glycogen concentration slightly decreased in the liver of cows in each treatment group from d 14 prepartum to d 5 postpartum; however, this decrease was more intensive in both the control and CAS groups than in the HTG group. The variations in liver lipid concentrations were accompanied by significant changes in the proportion of C16:0, C16:1n-7, C18:0, C18:1n-9, C18:2n-6 and C20:4n-6 fatty acids in the liver lipids. The results show that HTG supplementation exerted more advantageous effects on liver lipid and glycogen metabolism than did CAS supplementation.     

Effects of Increased Ambient Temperature During IVM and/or IVF on the In Vitro Development of Bovine Zygotes

Previous research by this group (2003) has demonstrated that heat stress during in vitro culture (IVC) significantly increased early embryo mortality. The experiments reported here examine the effects of heat treatment (HT) during in vitro maturation (IVM) and during in vitro fertilization (IVF). One 24 h cycle of HT entailed a series of 0.5 degrees C incubator temperature increases from 39 degrees C to 39.5 degrees C for 2 h, to 40 degrees C for 2 h, to 40.5 degrees C for 4 h, 41 degrees C for 4 h, 40.5 degrees C for 6 h and 40 degrees C for 6 h. This cycle mimics rectal temperatures recorded in high producing, grain fed dairy cows in hot climates. Experiment I studied the effects of one cycle of heat-treatment during IVF on the rate of cleavage of in vitro matured presumptive zygotes. Total cleavage rate in the HT group (37.8%) was lower than that of the control group (54.6%, p < 0.05). Experiment II repeated the HT of experiment I but preceded it with a cycle of HT during IVM. The total cleavage rates for control and heat treatment groups were 75.5% and 37.9%, respectively, with a significant difference of p < 0.001 identified. Experiment III examined the rates of embryonic development to >or=8-cell stage (after 72 h IVC) and to morula or blastocyst (M/B) stage (after 144 h IVC) following HT of the oocyte groups during the preceding IVM or IVF. Rates of development to >or=8-cell stage (at 72 h IVC) and to M/B stage (after 144 h IVC) for the control group were 27.5% and 35.8%. Those of IVM-only HT and IVF-only HT groups were 13.8% and 14.6%, and 8.6% and 14.3%, respectively. Both groups of heat treated embryos developed at significantly lower rates (p < 0.05) than did the control group. These results suggest that hyperthermia during oocyte maturation and/or fertilization adversely affects oocyte maturation and fertilization rates and retards further embryonic development.