Effects of β-OH Butyrate on Bovine Granulosa and Theca Cell Function in Vitro

In this study, the effects of beta-OH butyrate (BHB) levels, associated with a negative energy balance, on bovine granulosa and theca cell function were investigated in vitro. Granulosa and theca cells of healthy large follicles (>8 mm), obtained from slaughterhouse ovaries, were cultured in serum free medium containing 0, 0.5, 1 or 1.5 mm BHB and 3 mm glucose, to mimic the situation in the early postpartum dairy cow. Hormone concentrations (progesterone, oestradiol-17beta and/or androstenedione) in spent medium and cell numbers were measured after 48 h of culture. No effects of BHB on theca cell numbers or on steroid production were observed. In granulosa cells, all BHB treatments evenly increased cell numbers (p < 0.05), while they reduced progesterone and oestradiol-17beta production per cell (p < 0.05). These effects may be attributed to the use of BHB as energy source which is however differently metabolized than glucose. Conclusively, in the presence of physiological glucose concentrations BHB can modulate granulosa but not theca cell function in vitro.     

Comparative IFN-τ Secretion after Hatching by Bovine Blastocysts Derived Ex Vivo and Completely Produced In Vitro

The interferon-tau (IFN-tau) secretion levels after hatching by bovine blastocysts derived from in vitro maturated oocytes (Group A) and from in vivo (Group B) were investigated considering embryo quality. Only very homogeneous blastocysts of excellent or good quality were considered from day 7 of culture (Group A) and day 7 after artificial insemination with frozen-thawed from the same bull used for in vitro fertilization (Group B). All embryos were individually cultured into a 50 microl droplet of synthetic oviduct fluid medium with 10% fetal calf serum. After 24-h culture both Group A (n=44) and B (n=40) secreted <54 pm IFN-tau. After 48-, 72-, 96- and 120-h culture, Group A daily secreted 143 +/- 24 pm IFN-tau (n=19) vs 85 +/- 12 pm IFN-tau (n=21) for Group B (p < 0.01), 491 +/- 128 pm IFN-tau (n=29) vs 216 +/- 37 pm IFN-tau (n=23) (NS), 499 +/- 135 pm IFN-tau (n=26) vs 353 +/- 93 pm IFN-tau (n=21) (NS), 559 +/- 136 pm IFN-tau (n=22) vs 333 +/- 75 pm IFN-tau (n=20) (NS), respectively. Taken all together during 5 days, Group A produced per embryo 1690 +/- 290 pm IFN-tau (n=22) vs 982 +/- 182 pm IFN-tau (n=20) for Group B (p < 0.05). For all culture time there were sizable percentages of embryos that did not produce concentrations of IFN-tau above a certain cut-off level, and as such were not used to compute the means. In respect of the embryo quality whatever the groups after days 7-12 of culture, IFN-tau secretions were 1815 +/- 453 pm (n=10) for the embryos of excellent quality vs 1356 +/- 200 pm (n=28) for those of good quality (NS) and 360 +/- 188 pm (n=4) (p < 0.05) for embryos of fair quality. A positive relationship between IFN-tau production and in vitro development of quality I embryos was observed, whatever the embryos origins and, the embryos completely produced in vitro secreted more IFN-tau than the embryos produced in vivo.     

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.     

Cystic Degeneration in Porcine Ovaries - Second Communication: Concentrations of Progesterone, Estradiol-17β, and Testosterone in Cystic Fluid and Plasma; Interpretation of the Results

Contents: The content of progesterone, estradiol-17β, and testosterone of plasma and cystic fluid was determined in 79 sows with ovarian cysts. The average progesterone concentration of sows with dark corpora lutea (CL) was higher than of sows with pale or absent CL (39.4 vs. 8.7 vs. 8.0 ng/ml plasma; p < 0.001; and 7512 vs. 3644 vs. 2723 ng/ml cystic fluid, respectively; p < 0.001). The cystic fluid of animals with oligocystic ovaries (10 cystslanimal) had a significant higher progesterone concentration in comparison to potycystic animals (7200 vs. 3682 ng/ml; p < 0.001). Testosterone and estradwl-17β levels in plasma and in cystic fluid of polycystic animals were significantly higher in comparison to oligocystic animals (Plasma-Testosterone: p < 0.01; Plasma-Estradwl: p < 0.05; Cyst-Testosterone: p < 0.01; Cyst-Estradiol: p < 0.001). In oligocystic ovaries testosterone in cysts exceeded the estradiol-17β levels, whereas in polycystic ovaries the situation was vice versa (p < 0.001).
It is suggested that cystic ovarian degeneration in the sow is not exclusively a gradually progressing process, rather then a complex syndrome with three components, were characterized by a separate course of development (oligocystic, polycystic. oligo-polycystic syndrome).     

Influence of Cysteamine on In Vitro Maturation, In Vitro and In Vivo Fertilization of Equine Oocytes

The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, IVF or in vivo embryonic development under our conditions. Ovum pick up yields (52%) and maturation rates (control group: 47% and cysteamine group: 55%) were similar to those previously reported. From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and five in the cysteamine group (9%). Those two percentages were not statistically different (p > 0.05). No effect of IVF technique was seen on the success rate (6%) in each group.     

牛体外受精胚胎的体外培养研究进展

家畜体外受精（IVF）技术自上世纪八十年代获得成功以来，发展十分迅速，九十年代已进入试管胚的开发应用阶段。目前，IVF胚胎的质量与体内胚相比仍有很大差距。在进行IVF胚胎体外培养时，常会出现“体外发育阻断”现象，主要表现为细胞数较少、卵裂球形状不规则、存在死亡的细胞和细胞质碎片、发育速率和抗冻性降低以及酶活性和营养物摄取的改变等方面。近年来，为了克服胚胎体外发育所存在的某些缺陷，国内、外对不同动物的胚胎体外培养体系和培养方法进行了系统研究，取得了长足进展。本文就牛IVF胚胎体外培养方面的研究进展综述如下。     

Viability and Growth of Buffalo Preantral Follicles and their Corresponding Oocytes In Vitro: Effect of Growth Factors and β Mercaptoethanol

The present study was undertaken to isolate buffalo preantral follicles (PFs), to test the viability and sizes of buffalo PFs and to examine the effect of various growth factors (insulin-like growth factor, fibroblast growth factor) and an antioxidant (β mercaptoethanol) on the in vitro growth, survival and antrum formation rates of buffalo PFs and growth rates of oocytes in cultured PFs. Preantral follicles from slaughtered buffalo ovaries were recovered by a combined mechanical and enzymatic method. The recovery rates of >40–100, 101–200, 201–300, 301–400 and 401–500 μm PFs were 5.1, 3.2, 3.1, 6.3 and 5.1 per ovary, respectively. The corresponding viability rates were 76.1%, 78.1%, 85.2%, 92.5% and 92.6%, respectively. There was a positive correlation ( r  = 0.73) between oocyte size and the follicular size. However, there was no significant correlation between the size of oocyte and its viability at the time of its retrieval from ovary. Insulin-like growth factor and fibroblast growth factor improved the survival of buffalo PFs and regulated their growth in culture. The growth factors and β mercaptoethanol in association synergically improved the growth and survival of buffalo PFs.     

Effect of Oestrous Synchronization with Estradiol 17β and Progesterone on Follicular Wave Dynamics in Dairy Heifers

An experiment was designed to evaluate the effects of estradiol‐17β (E17β) on follicular wave dynamics and ovulatory response in Holstein heifers receiving either a progestogen ear‐implant (Crestar^®; Intervet International b.v. Boxmeer, The Netherlands) or an intravaginal progesterone‐releasing device [controlled internal drug release‐bovine device (Eazibreed, CIDR‐B^®; Bodinco BV, Alkmaar, The Netherlands)]. For comparison, another group of heifers was also synchronized using Crestar plus an injection of estradiol valerate (EV) and norgestomet as recommended by the pharmaceutical company. Twenty 20–22‐month‐old cycling Holstein heifers were allocated to one of the following treatment groups at random stages of the oestrous cycle: (I) simultaneous insertion of Crestar and intramuscular injection of 3 mg norgestomet and 5 mg EV (Crestar 9 + EV 9); (II) simultaneous insertion of Crestar and intramuscular injection of 5 mg E17β (Crestar 9 + E17β 9); (III) insertion of Crestar followed 2 days later by intramuscular injection of 5 mg E17β (Crestar 9 + E17β 7); or (IV) insertion of CIDR‐B device followed 2 days later by intramuscular injection of 5 mg E17β (CIDR 9 + E17β 7). The CIDR‐B or Crestar implants were removed after 9 days and all heifers received 500 μg Cloprostenol (Estrumate^®, Pitman‐Moore Nederland BV, Houten, The Netherlands). Ovarian ultrasonographic examinations were performed once daily during the synchronization period using a B‐mode scanner equipped with a 7.5 MHz linear‐array transrectal transducer. In addition, heifers were scanned every 12 h after implant/device withdrawal until 3 days after ovulation in order to monitor follicular activity, detect ovulation and subsequent early luteal formation. Detection of oestrus was performed every 6 h for 4 days after device/implant removal. Oestrus was observed 24–32 h before ovulation in all heifers. The mean hours interval from treatment withdrawal to ovulation was not significantly different (84.0 ± 16.5, 77.6 ± 4.1, 73.6 ± 4.1 and 64.0 ± 4.4 h for treatments I, II, III and IV, respectively; p > 0.1). However, the variance for heifers treated with EV + norgestomet was significantly larger (Levene’s Test; p < 0.01) than those treated with E17β. All E17β treatments resulted in dominant follicle suppression and a new wave emerged 4.1 days after treatment compared with 6.6 days for the EV + norgestomet treatment (p < 0.05). The time from emergence of the new ovulatory wave to ovulation was longer for the new wave that emerged after E17β treatment (9.2 ± 0.3 days) than after EV + norgestomet treatment (6.9 ± 0.4 days; p < 0.05). The results of this study suggest that the four treatments used were effective in inducing synchronous behavioural oestrus and ovulation. However, a higher degree of oestrus and ovulation synchrony was observed in heifers treated with E17β than in heifers treated with EV + norgestomet. Synchronization treatments with exogenous E17β or EV + norgestomet at the time of progestin device insertion (Crestar or CIDR‐B) or 2 days later in heifers can regulate a different emergence pattern of ovarian follicular development in randomly cyclic heifers. The E17β was effective in inducing follicular suppression and resulted in the consistent emergence of a new follicular wave.     

Occurrence and Importance of Glomus Organs (Hoyer-Grosser's Organs) in the Skin of the Equine and Bovine Mammary Gland

Glomus organs (Hoyer-Grosser's organs) were frequently found in the corium and the subcutis of the skin of the equine and bovine mammary gland. They were most frequently situated in the border zone between the stratum profundum and the stratum superficiale corii. These specialized vascular structures (arterio-venous anastomosis) were present in all investigated skin areas. Although the glomus organs varied in size and shape, they possessed common histologic structures: an arteriole entered the connective capsule of the glomus and divided into strongly convoluted arterio-venous channels; the arterio-venous channels united in the end to form a venule; the mentioned vascular elements were covered by a connective capsule and were thus united to an organ-like structure. Questions concerning their occurrence, their functional interpretation, their relevance, the size of the glomus organs as well as the possible involvement of the lymphatics were discussed.     

生物频谱对牛胚胎体外生产效率的影响

本试验利用可调式周林生物频谱发生器对牛卵母细胞体外成熟、体外受精和体外培养过程实施辐射 ,以提高牛胚胎体外生产效率。频谱发生器与培养材料的距离为 5cm ,辐射强度根据辐射温度加以调节。实验结果表明 ,辐射温度控制在 39℃时 ,入孵卵母细胞的卵裂率 ( 63% )与对照组 ( 61 % )无显著差异(P >0 0 5) ,而囊胚发育率 ( 39% )显著高于对照组 ( 1 9% ) (P <0 0 5) ;当辐射温度达到 4 0℃时 ,实验组的卵裂率仅为 1 4 % ,囊胚率为 0。体外受精生产的囊胚用EFS4 0液进行玻璃化冷冻保存 ,解冻后发现 ,整个生产过程经生物频谱辐射胚胎的体外发育率 ( 86% )显著高于对照组 ( 65% )。     

饲粮添加精氨酸对娩后Wistar大鼠乳腺组织发育及酪蛋白合成的影响

本试验旨在研究饲粮添加精氨酸(Arg)对娩后Wistar大鼠乳腺组织发育及其酪蛋白合成的影响。采用同窝对照的方法,选择同窝12只已受孕的Wistar大鼠,随机平均分为2组。Arg组在基础饲粮中添加与其等量的Arg(饲粮Arg含量1.280%),对照组饲粮用谷氨酸替代Arg组中的Arg以平衡饲粮氮水平。预试期4 d(分娩前4天)和正试期17 d(分娩后17天),结束后采集泌乳大鼠的乳腺组织,制作组织切片并测定其酪蛋白含量。结果表明:Arg能促进乳腺组织的发育,且乳腺腺泡切面面积的极显著增大(P0.01)。另外,Arg对乳腺β-酪蛋白的含量有显著的提高作用(P0.05),同时娩后大鼠体重也有增加的趋势(P0.05)。结果提示,饲粮添加Arg对Wistar大鼠乳腺组织发育及β-酪蛋白的合成有显著的促进作用。     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

体外法研究竹叶黄酮对奶牛乳腺上皮细胞抗氧化及哺乳动物雷帕霉素靶蛋白信号通路中关键基因表达的影响

本试验旨在通过体外法研究竹叶黄酮(BLF)对奶牛乳腺上皮细胞(BMECs)抗氧化及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路中关键基因表达的影响.采用单因素试验设计,对照组不添加BLF,试验组分别添加不同浓度(0.5、1.0、5.0、10.0、20.0、60.0、100.0μg/mL)BLF,处理BMECs 3、6、1...