The study aimed to compare differences of physiological acid-base balance (ABB) parameters in follicular fluid (FF) and venous blood (VB) and to evaluate ABB parameters in FF collected from different ovarian follicles in dairy cows and heifers. The ABB parameters (pH, pCO2, pO2, HCO3– and base excess (BE)) in the FF of the preovulatory follicle, of the dominant follicle on the 9th day of the cycle and of the superovulatory estrous follicles were compared to VB. Similarly, the dynamics of the ABB profile in FF and VB were monitored in repeated sampling in a group of heifers stimulated by follicle-stimulating hormone (FSH). Higher values of pH and pO2 and lower values of pCO2, HCO3– and BE were found in FF compared to VB in all experiments. Laterality of ovaries, time of sampling, ovarian activity or stimulation of the follicular development by FSH did not significantly influence ABB parameters. We found higher pH (7.392 ± 0.027 vs. 7.364 ± 0.032) and pO2 (13.83 ± 2.20 kPa vs. 4.50 ± 0.67 kPa), lower pCO2 (5.70 ± 0.39 kPa vs. 6.54 ± 0.61 kPa), HCO3– (25.51 ± 1.52 mmol/l vs. 26.86 ± 2.12 mmol/l) and BE (1.14 ± 1.57 mmol/l vs. 1.95 ± 2.2 mmol/l) in FF compared to VB in all non-stimulated cows. Similar relationships between FF and VB were found in all FSH stimulated cows. The study provides as yet unknown knowledge on the physiology of follicular fluid in cattle.

The effect of docosahexaenoic acid (DHA) on ovarian activity of dairy cows was determined. Experimental cows (n = 25) were fed a total mixed ration supplemented daily with 100 g/cow of an algae product All-G-Rich (Alltech, Ireland) containing 10% DHA divided into 2 doses for 52 days. Determination of DHA from milk samples taken from all cows was performed before the All-G-Rich supplementation (on Day 0, D0), and on D21 and D42 of algae supplementation. Cows were synchronized to be in oestrus on D21 and D42 of the experimental period. Monitoring of ovarian activity was performed by transrectal ultrasonography. Examinations were performed at 2-3-day intervals from D0 until D52 of the experimental period. Plasma concentrations of progesterone, oestradiol, insulin, NEFA, and cholesterol were determined. Control cows (n = 25) were examined in the same way as the experimental cows. Milk DHA concentrations on D21 and D42 were significantly higher in treated cows (D21 1.38 vs 0.28, P < 0.0001; D42 1.34 vs 0.20, P < 0.0001). There were neither important effects of DHA on ovarian structures, nor on evaluated variables in plasma. Cows in the experimental group tended to have larger corpora lutea and higher cholesterol concentrations, but differences were not significant.

This study evaluated the occurrence of two corpora lutea (CL) and the pregnancy rate in cows treated with gonadorelin on days 5, 6 or 7 after insemination in relation to the day of treatment and the size of the largest follicle on the ovaries at the time of treatment. Cows bearing one CL on days 5 (n = 40), 6 (n = 48), and 7(n = 47) after insemination with defined size of the largest follicle on the ovaries (small = 9 mm, n = 34; medium 10-14 mm, n = 59; large 15-20 mm, n = 42) were included in the study. Gonadorelin was administered after examination (day 0) and ultrasonographic examination was repeated on days 14, 28, and around day 90. The occurrence of 2 CL vs 1 CL was higher in pregnant cows on day 14 as well as on day 28 (86 vs 14% and 82.5 vs 14%, n = 57). Pregnancy rates on day 28 and around day 90 were higher in cows bearing 2 CL (n = 57) vs cows bearing 1 CL (n = 54) (82.5 vs 18.5% and 79.0 vs 18.5%, P < 0.001). The occurrence of 2 CL on day 28 was higher in cows treated on day 5 compared to cows treated on day 6 or 7 after insemination (60.0 vs 33.3 or 36.2%, P < 0.05). No significant differences in the number of CL and pregnancy rate were found in relation to follicle size at the time of treatment. Our results showed the higher efficiency of accessory CL induction by gonadorelin administration in cows treated on day 5 vs those treated on day 6 or 7 post insemination, however efficiency of the treatment was not influenced by the size of the largest follicle on the ovaries.

Acid-base balance is one of the most vigorously regulated variables of the body, including genital organs. Subacute ruminal acidosis is a common disturbance in dairy cows that disturbs several biochemical indices in the blood, cerebrospinal fluid, and urine. The possible negative effects of metabolic acidosis on the follicular fluid (FF) composition and, subsequently, on oocyte quality, are not fully elucidated. This study aimed to evaluate the changes in acid-base balance (ABB) in FF and blood during acute metabolic acidosis in dairy heifers. Ten Holstein heifers were stimulated with FSH in eight decreasing doses at 12-hour intervals (D0-D3). Acidosis was induced by oral administration of sucrose at 9 g/kg of body weight, dissolved in 10 L of warm tap water, at D3. Samples were collected from each cow at 0, 8, 12, 16, 24, 32, 40, and 48 hours after treatment. Samples of FF, obtained by transvaginal follicular aspiration, and peripheral blood were examined for ABB parameters: pH, pCO2, pO2, HCO3-, and base excess (BE). A significant decrease in pH, HCO3-, and BE values in the blood, as well as FF, occurred after sucrose treatment. The lowest pH values occurred in blood at 16 hours, and in FF at 24 hours, after treatment (7.30 ± 0.05 and 7.33 ± 0.05, respectively). The lowest HCO3- values in blood (18.75 ± 3.2 mmol/L) and FF (18.07 ± 2.84 mmol/L) occurred 24 hours after treatment, as did the lowest BE values (-6.61 ± 3.7 mmol/L and -7.53 ± 3.89 mmol/L, in blood and FF, respectively). Significant correlations for HCO3- (r = 0.928), BE (r = 0.946), pH (r = 0.889), and pCO2 (r = 0.522) existed between blood and FF samples. The results demonstrated that metabolic acute acidosis substantially influences the characteristics of both serum and FF.

The aim of this study was to evaluate the effects of administration of oxytocin, in combination with dinoprost or ergometrin, after parturition on the occurrence of clinical endometritis and subsequent reproductive performance in dairy cows. The cows, without retained foetal membranes, were assigned to one of three groups. No treatment was performed in the control group (Group C, n = 229). However, intramuscular administrations of oxytocin (30 IU) on Days 1 and 2 post partum, in combination with dinoprost (25 mg, Group D, n = 123) or ergometrin (15 mg, Group E, n = 130) administered on Day 7 post partum, were performed in experimental groups. Vaginal discharge score (VDS, 1-5), uterine content score (UCS, 1-3) on Day14 to 21 post partum, number of treatment interventions (TI), proportion of non-treated cows (NT) because of endometritis, calving to first insemination interval (CI), first insemination conception rate (CR), and proportion of pregnant cows by Day 150 post partum (PC), were compared among groups. There was no significant difference among groups for any parameter. Average values were 2.08, 2.24 and 2.06 (VDS); 1.49, 1.51 and 1.45 (UCS); 0.86, 0.93 and 0.74 (TI); 59.4, 54.2 and 54.7% (NT); 73.9 ± 15.2, 75.4 ± 17 and 74.2 ± 17 days (CI); 41.4, 38.9 and 44.3% (CR); and 75.4, 77.7 and 80.5% (PC) for Groups C, D and E, respectively. There was no significant benefit of preventive administration of oxytocin, in combination with dinoprost or ergometrin, after parturition on reproductive performance in dairy cows.

Sufficiency of progesterone limits conception rate in dairy cows. This study evaluates the effect of gonadotropin-releasing hormone on induction of accessory corpus luteum, progesterone concentrations and pregnancy in dairy cows. The hormone (0.05 mg) was administrated intramuscularly to cows with one corpus luteum on day 5–7 (Group E1, n = 57) or 11–13 (Group E2, n = 40) after insemination; control groups C1 (n = 54) and C2 (n = 41) were not treated. Detection of pregnancy by ultrasound examination and measurement of serum progesterone concentration was performed on day 25–39 and around day 90 after insemination. The occurrences of two corpora lutea in groups E1 and E2 versus C1 and C2 were 43.9% and 33.5% vs. 3.7% and 9.8% (P < 0.001). Progesterone concentrations were 5.9 ± 2.92 ng/ml and 7.8 ± 2.35 ng/ml in untreated and treated cows (P = 0.016), respectively. Conception rates in groups E1 and E2 were 100% and 86.7% in cows bearing two corpora lutea, and only 16.7% and 38.9% in cows bearing one corpus luteum (P < 0.01); similarly as in groups E1 and E2 compared to groups C1 and C2 (49.1% and 50% vs. 50% and 31.7%). Pregnancy rates around day 90 in groups E1 and E2 compared to groups C1 and C2 were 45.6% and 45% vs. 42.6% and 26.8%, respectively. Administration of gonadotropin-releasing hormone on day 5–7 or 11–13 after insemination was efficient to induce accessory corpus luteum and increase the concentration of serum progesterone but had no effect on the conception rate.

The aim of this study was to evaluate the effect of intrafollicular treatment (IFT) with different doses of luteinising hormone. Experimental heifers were treated with a single deslorelin implant to desensitise gonadotroph cells of the pituitary gland. Thereafter, follicular development was stimulated by exogenous FSH treatment. Intrafollicular treatment with 10, 5, 1 and 0.01 µg LH was performed on one single follicle while other follicles remained untreated. Human chorionic gonadotrophine (2000 UI) was administered intravenously as a control. Ovulation and development of the corpus luteum occurred after all intrafollicular treatments with 10 and 5 µg LH. After IFT using 1 µg of LH 75% animals (3/4) ovulated. The dose of 0.01 µg was not followed by any ovulation whereas control treatments with hCG were followed by an ovulation of the majority of follicles present in the ovaries. In conclusion, IFT with different doses of LH (greater than 0.01 µg) is capable of inducing ovulation.

The aim of this study was to evaluate a new device for the ultrasound-guided transvaginal aspiration of follicular fluid for acid-base balance analysis (ABB set) in comparison with the original modified commercial OPU set. In the ABB set, an aspiration syringe was placed in the front part of the new tool's handle, next to the transducer, so as to enable direct collection of the sample into the syringe. To obtain a sufficient amount of testable fluid, reservoirs of urine (rubber balloons) were used for later aspiration under laboratory conditions in Experiment 1. Fifteen triads of samples (each triad with two punctures) were collected. While the first sample of each triad was taken using the ABB set (ABB sample), two samples were taken by one puncture using the original modified commercial OPU set: aerobic phase of sampling (AE sample) with air present in the tubing at the start of sampling and the subsequent anaerobic phase of sampling (AN sample). Values determined in the second sample from the triad (AE) varied from the values in both ABB and AN samples (pH 7.685 vs. 7.704 vs. 7.692, pCO2 11.13 vs. 10.3 vs. 10.85, pO2 6.87 vs. 8.67 vs. 7.02). In Experiment 2, ultrasound-guided transvaginal aspirations were carried out in 13 cows bearing ovarian cysts with diameters of at least 3 cm, using plastic aspiration syringes (Experiment 2P) and in 12 cows using glass aspiration syringes (Experiment 2G). The sequence of samples was the same as in Experiment 1. We found a significantly higher pH in AE in comparison to AN (7.357 vs. 7.348), lower pCO2 (6.85) and higher O2 (14.12) in samples of AE in comparison to samples of ABB and AN (pCO2 7.36, 7.30; O2 9.95, 10.63 respectively) in cystic fluid in Experiment 2P. We found a significantly higher pH (7.4), lower pCO2 (5.98) and a higher pO2 (12.35) in AE samples in comparison to ABB and AN samples of cystic fluid (pH 7.386, 7.385; pCO2 6.39, 6.35 and O2 10.56, 10.65, respectively) from Experiment 2G. We conclude that the acid-base balance assay was affected by air, present in the tubing during aerobic sampling in comparison to anaerobic and ABB set sampling. These pre-analytical changes can be prevented by the use of the ABB set because the results obtained with the ABB set were not different from that of the AN samples. We also confirmed pre-analytical changes in acid-base balance parameters in the cystic fluid after it had been stored in plastic aspiration syringes. Our new ABB set equipped with a glass aspiration syringe is suitable for sampling follicular fluid for both acid-base balance and gas analysis.

The aim of this study was to evaluate a new double-channel system for ultrasound-guided transvaginal intrafollicular treatment in cattle. The system is equipped with separate aspiration and treatment channels facilitating the aspiration of a small part of follicular fluid followed by the immediate injection of the same amount of treatment solution. In Experiment 1 an intracystic injection was performed successfully in all cows (6/6). In Experiment 2 saline was administered to dominant follicles with an overall success rate of 87.5% (14/16). This new double-channel system represents a reliable method for intrafollicular treatment preceded by an aspiration of the necessary amount of follicular fluid without an increase in intrafollicular pressure.

The aim of this study was to determine the minimum effective intrafollicular doses of luteinizing hormone and human chorionic gonadotropin in order to induce ovulation in cycling dairy heifers that have not yet been adequately established. Application of 10, 5, 1, 0.5, 0.1, 0.01 and 0.001 µg luteinizing hormone as well as 10, 1, 0.1, 0.01 and 0.001 international units (IU) of human chorionic gonadotropin in dominant follicles was performed on day 7 of the oestrous cycle. Control animals were given luteinizing hormone (12.5 mg and 25 mg) or human chorionic gonadotropin (2000 IU) intravenously. Accessory corpus luteum on day 14 of the oestrous cycle was considered as an evidence of ovulation. Ovulation was observed in 2 out of 3 heifers in each treatment group (n = 3) after administration of 10–0.1 µg luteinizing hormone (except for 0.5 µg – ovulation in 3 of 3 heifers), in all heifers after administration of 10–0.01 IU human chorionic gonadotropin as well as in all control heifers. Administration of 0.01 µg and 0.001 µg luteinizing hormone as well as of 0.001 IU human chorionic gonadotropin did not result in ovulation. Higher progesterone concentration on day 14 vs. day 7 of the oestrous cycle was found after all treatments. Nevertheless, the differences were significant ( P < 0.05) only after intrafollicular treatments with 5, 1 and 0.001 µg luteinizing hormone as well as 10, 1 and 0.01 IU human chorionic gonadotropin. In conclusion, minimum efficient doses for intrafollicular treatment of the dominant follicles in cycling heifers capable of inducing ovulation were 0.1 µg of luteinizing hormone and 0.01 IU of human chorionic gonadotropin. This is the first study describing the intrafollicular luteinizing hormone administration in cycling dairy heifers.

The purpose of the trial was evaluation of follicular growth and endometrial folding in mares after human chorionic gonadotropin (hCG) treatment in comparison with untreated mares during oestrus. In addition, the influence of follicle size at the time of hCG treatment on these parameters was evaluated. HCG (3000 IU) was administered intravenously in 17 mares bearing dominant follicles 35-40 mm in diameter (Group A) and in 13 mares with larger follicles (Group B). Ten mares with follicles ≥ 35 mm were untreated (Group C). Ultrasonographical examination of the mares continued in 6 h intervals until ovulation. Growth of the dominant follicle was faster in Group A than in Groups B and C (1.3 vs. 0.3 and 0.7 mm/6h, P < 0.05) but diameters of the preovulatory follicles were similar - 44, 48 and 44 mm in Groups A, B and C, respectively. Similarly, reduction of endometrial folding (on a three point scale) during observation was higher in Group A than in B and C (2.1 vs. 1.2 and 1.8, A : B P < 0.05) but endometrial folding values in the term before ovulation were not different (0.6, 0.9 and 0.6 in Groups A, B and C). A positive correlation between the speed of follicular growth and reduction of endometrial folding was found (rs - 0.479, P = 0.003). Irregularity in follicle shape (the difference between the longest axis and its perpendicular axis) at the beginning of observation (3.3, 4.0 and 3.2 mm) was lower than before ovulation (7.4, 10.4 and 9.2 mm) in all groups (P < 0.01). The interval from the beginning of observation until ovulation was significantly shorter in Groups A and B versus C (37 and 31 vs. 103 h, P < 0.01). The results show that growth of dominant follicles after hCG is influenced by the size of the follicles at the time of treatment and correlates with reduction in endometrial folding as well as irregularity of follicle shape. Nevertheless, hCG treatment does not influence the size and shape of preovulatory follicles or endometrial folding immediately before ovulation.

Pre-implantation embryos derived by in vitro fertilization differ in their developmental potential from embryos obtained in vivo. In order to characterize changes in gene expression profiles caused by in vitro culture environment, we employed microarray constructed from bovine oocyte and preimplantation embryo-specific cDNAs (BlueChip, Université Laval, Québec). The analysis revealed changes in the level of 134 transcripts between in vitro derived (cultured in COOK BVC/BVB media) and in vivo derived 4-cell stage embryos and 97 transcripts were differentially expressed between 8-cell stage in vitro and in vivo embryos. The expression profiles of 7 selected transcripts (BUB3, CUL1, FBL, NOLC1, PCAF, GABPA and CNOT4) were studied in detail. We have identified a switch from Cullin 1-like transcript variant 1 to Cullin 1 transcript variant 3 (UniGene IDs BT.36789 and BT.6490, respectively) expressions around the time of bovine major gene activation (8-cell stage). New fibrillarin protein was detected by immunofluorescence already in early 8-cell stage and this detection correlated with increased level of fibrillarin mRNA. The qRT-PCR analysis revealed significant differences in the level of BUB3, NOLC1, PCAF, GABPA and CNOT4 gene transcripts between in vivo derived (IVD) and in vitro produced (IVP) embryos in late 8-cell stage. The combination of these genes represents a suitable tool for addressing questions concerning normal IVD embryo development and can be potentially useful as a marker of embryo quality in future attempts to optimize in vitro culture conditions.

The aim of this study was to evaluate the applicability of ovum pick-up equipment for follicular fluid collection from various follicular structures (experiment 1) and for recovery of follicular fluid for acid-base balance analysis (experiment 2). An ultrasound scanner equipped with a 5-MHz convex transducer was used for transvaginal ultrasound-guided follicular aspiration. A 17-gauge, 60-cm aspiration needle was connected with a shortened aspiration line. The fluid was aspirated manually into a 2 ml plastic syringe at a speed of approximately 0.2 ml/s. The success of aspiration was higher in ovarian cysts (100%) and single follicles larger than 13 mm (76.7%) compared to single follicles smaller than 12 mm (20%,p< 0.001). The success of aspiration of multiple follicles on day 4 (diameter of 7–9 mm) was higher (90.9%) compared to follicles on day 2 (diameter of 4-6 mm) (66.7%,p< 0.05) in experiment 1. The fluid from ovarian cysts > 25 mm in diameter was aspirated in a two-step procedure (samples 1 and 2) for the determination of pH, HCO3,BE, pCO2and pO2(experiment 2). The indicators were compared between samples 1 and 2. Higher pO2as well as pH and lower pCO2in sample 1 compared to sample 2 showed insufficient anaerobic conditions during the first phase of the puncture in experiment 2. Our study brings for the first time the finding that the ovum pick-up equipment used in our experiments is suitable for the collection of follicular fluid only from larger follicular structures. The sampling of follicular fluid for acid-base balance assays requires the development of a special new device to prevent samples from coming into contact with air during aspiration.