Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality.

Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality.

Frozen-thawed ampullary cell monolayer improves bovine embryo in vitro development and quality.

The aim of this study was to evaluate the effects of different timing for frozen-thawed bovine ampullary epithelial cell (BAEC) and bovine oviductal epithelial cell (BOEC) co-culture on the development and quality of bovine embryos produced in vitro. Embryo development was assessed by day 8 blastocyst yield, whereas embryo quality was determined using blastocyst differential cell count, cryotolerance and the expression of selected genes related to embryo quality. The results showed that the presence of BAECs during the last 6 h of in vitro maturation (IVM) increased blastocyst yield and survival of the vitrified-warmed blastocysts. In addition, embryos produced in the presence of BAECs during the last 6 h of IVM or in the presence of BOECs during the first 4 days of in vitro culture (IVC) showed a greater number of trophectoderm cells and a greater inner cell mass. In terms of gene expression, IFN-T was downregulated and PLAC8, AQP3 and ATP1A1 were upregulated in the presence of the BAECs during the last 6 h of the IVM and/or in the presence of BOECs during the first 4 days of IVC. In conclusion, co-culturing bovine oocytes with a frozen-thawed ampullary cell monolayer during the last 6 h of maturation increased blastocyst yield and quality.

Prevalence, risk factors and consequent effect of dystocia in holstein dairy cows in iran.

The objective of this research was to determine the prevalence, risk factors and consequent effect of dystocia on lactation performance in Holstein dairy cows in Iran. The data set consisted of 55,577 calving records on 30,879 Holstein cows in 30 dairy herds for the period March 2000 to April 2009. Factors affecting dystocia were analyzed using multivariable logistic regression models through the maximum likelihood method in the GENMOD procedure. The effect of dystocia on lactation performance and factors affecting calf birth weight were analyzed using mixed linear model in the MIXED procedure. The average incidence of dystocia was 10.8% and the mean (SD) calf birth weight was 42.13 (5.42) kg. Primiparous cows had calves with lower body weight and were more likely to require assistance at parturition (p<0.05). Female calves had lower body weight, and had a lower odds ratio for dystocia than male calves (p<0.05). Twins had lower birth weight, and had a higher odds ratio for dystocia than singletons (p<0.05). Cows which gave birth to a calf with higher weight at birth experienced more calving difficulty (OR (95% CI) = 1.1(1.08-1.11). Total 305-d milk, fat and protein yield was 135 (23), 3.16 (0.80) and 6.52 (1.01) kg less, in cows that experienced dystocia at calving compared with those that did not (p<0.05).

Association between age at first calving and lactation performance, lactation curve, calving interval, calf birth weight, and dystocia in Holstein dairy cows.

In the present study, records on 115,291 heifers distributed in 113 herds were used to investigate the association between age at the first calving (AFC) and lactation performance, lactation curve, the length of the first calving interval (CI), calf birth weight (CBW), and the incidence of dystocia in Holstein heifers in Iran. Based on the AFC, the heifers were classified into eight classes: AFC of 541 to 690 d, 691 to 720 d, 721 to 750 d, 751 to 780 d, 781 to 810 d, 811 to 840 d, 841 to 900 d, and 901 to 1200 d (AFC1 to AFC8, respectively). Multiple regression mixed models were used to investigate the association between AFC and lactation curve parameters, partial and 305-d lactation performance, 100- and 305-d SCS, and the length of the first calving (CI) interval. The mean (SD) and median AFC across all heifers was 760.2 (74.01) and 750 d, respectively. Of 115,291 heifers included, 28,192 and 7,602 heifers were, respectively, ≤ 720 and > 900 d when calving for the first time. More than 44% of the heifers were at 691 to 750 d (23 to 25 months) of age when calving for the first time. An increased AFC was associated with increased partial and 305-d lactation performance, 100- and 305-d SCS, initial milk yield, milk production at the peak of lactation, upward and downward slopes of the lactation curve. The 305-d fat percentage was associated with AFC; however, there was no association between AFC and 305-d protein percentage. An increased AFC was also associated with decreased milk production persistency, delayed peak time, longer CI, and higher calf birth weight. Compared to heifers calving for the first time between 691 to 780 d (23 to 26 months) of age, both increasing and decreasing AFC were associated with increased risk of dystocia. Controlling AFC is an important management factor in achieving a lower risk of dystocia, higher lactation performance, lower SCS, and shorter length of the calving interval.

Bovine oocyte developmental competence and gene expression following co-culturing with ampullary cells: An experimental study.

BACKGROUND: There is no sufficient information on the impact of bovine ampullary oviductal epithelial cells (BAOECs) on in vitro oocyte maturation competence and gene expression. OBJECTIVE: This study aimed to examine the oocyte developmental competence following co-culturing with a monolayer of fresh and frozen-thawed ampullary cells. MATERIALS AND METHODS: Bovine cumulus-oocyte complexes (COCs) were distributed into three groups: control group; where in COCs were cultured in cell-free media for 24 hr and FML and FTML groups in which the COCs were cultured in maturation media for 18 hr and then transferred into a media containing fresh and frozen-thawed BAOECs monolayer, respectively (BAOECs were extracted from the oviducts of slaughtered cattle and were then cultured freshly or frozen-thawed) for a further 6 hr. After 24 hr, the expanded COCs were evaluated for nuclear maturation, fertilization rate, and gene expression (GDF9, StAR, CASP3, and FSHr). RESULTS: Nuclear maturation rate in the FTML group was significantly higher than the control group (p = 0.02). The fertilization rate of FTML group was significantly higher than the control and FML groups (p = 0.05 and p = 0.03, respectively). In terms of gene expression, GDF9 were upregulated in the presence of the BAOECs during the last 6 hr of the in vitro maturation (p < 0.001). Furthermore, the expression of the StAR gene in the FTML group was higher than the other groups (p = 0.02). CONCLUSION: Ampullary cells co-culturing (especially frozen-thawed cells) for in vitro maturation of bovine oocytes yields encourages the results and demonstrates the beneficial effect of co-culture on gene expression and developmental competence.

Follicular fluid during individual oocyte maturation enhances cumulus expansion and improves embryo development and quality in a dose-specific manner.

We evaluated the effect of supplementation of different concentrations of bovine follicular fluid (FF) during in vitro maturation (IVM) on oocyte development and blastocyst quality in group and individual culture conditions. To do so, in vitro maturation medium (TCM-199 with 20 ng/mL epidermal growth factor and 50 µg/mL gentamycin) was supplemented with 0 (control), 1, 5, or 10% of FF. Follicular fluid was collected from slaughterhouse-derived ovaries, selecting follicles between 12 and 20 mm in diameter. Oocytes were either produced in groups or individually matured, fertilized, and cultured to the blastocyst stage, allowing for separate follow-up of each oocyte. Development (cleavage and blastocyst rates) among experimental groups were fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. We also assessed the cumulus expansion (prior and after maturation) for individual culture conditions, and their difference was fitted in mixed linear regression models. The FF was collected from two batches, with an estradiol/progesterone ratio higher than 1. The FF batch did not affect the development or blastocyst quality in group or individual culture conditions (P > 0.05). In group culture, development was similar among experimental groups (P > 0.05). Five or 10% of FF supplementation improved (P ˂ 0.05) aspects of blastocyst quality such as total cell numbers (TCN), trophectoderm (TE), inner cell mass (ICM), and ICM/TCN and apoptotic cells/TCN ratio in comparison to control. In the individual culture system, 5% FF supplementation increased (P ˂ 0.05) day 8 blastocyst rate (33 ± 3.4% (LSM ± SE)) in comparison to control (20 ± 2.7%) and 1% FF supplementation (19 ± 2.6%) but it was not different (P > 0.05) from 10% FF supplementation (28 ± 3.4%). Five percent of FF supplementation resulted in greater TCN, ICM, and ICM/TCN than control (P ˂ 0.05). It also resulted in a greater expansion of cumulus cell investment than the other groups (P ˂ 0.05), with a 3-fold increase compared to control. In conclusion, 5% of FF supplementation during IVM improved the cumulus expansion and the blastocyst development and quality in an individual culture system. However, FF supplementation during maturation in a group culture system did not increase development, but it modestly improved some embryo quality aspects when 5 or 10% of FF was added.

Association between gestation length and lactation performance, lactation curve, calf birth weight and dystocia in Holstein dairy cows in Iran.

In this study, 252,798 lactations on 108,077 cows in 433 herds were used to determine the association between gestation length (GL) and lactation performance, lactation curve, calf birth weight and dystocia in Holstein dairy cows in Iran. The GL averaged 278.1 ± 5.41 d, was categorized as short (SGL; at 1 SD below the population mean), average (AGL; the population mean ± 1 SD), or long (LGL; at least 1 SD above the population mean). Factors including parity, calf gender and calving season were associated with the GL. Primiparous cows with SGL had less lactation performance than those with longer GL; however, there was no difference between those with AGL and LGL. Multiparous cows with longer GL always had more partial and 305-d lactation performance. Primiparous cows with SGL produced less milk at the beginning of lactation and at the peak than those with AGL or LGL; inverse trends were found for lactation persistency, upward and downward slopes of the lactation curve. Within multiparous, a direct relationship was found between GL and the peak yield, where cows with longer GL always produced more milk at the peak. Multiparous cows with SGL produced less milk at the beginning of lactation, reached their peaks later, had higher lactation persistency and showed a lower upward slope of lactation curve than those with AGL or LGL. There was a direct relationship between GL and calf birth weight, where cows with longer GL had calves with more weight at the birth. Within primiparous, cows with SGL had the lowest and those with LGL had the highest rate of dystocia. However, multiparous cows with AGL had a lower rate of dystocia than those with SGL or LGL. Although there was a direct relationship between GL and lactation performance, intermediate GL seems optimal when considering dystocia.