A perfusion system for antibody production by shear-sensitive hybridoma cells in a stirred reactor

Summary A shear-sensitive hybridoma cell line, incapable of growth or antibody production in spinner or shake flasks agitated at 40 rpm, was grown successfully in a perfusion propagation system consisting of a bioreactor (1.5 liter), stirred with a cell-lift impeller at 60 rpm, and a tangential flow filtration unit for removal of spent culture medium from the reactor. The culture was maintained over a 48 day period and cell numbers reached 1.8 × 10⁷ cells/ml. Maximal monoclonal antibody concentration was 800 ug/ml, indicating a productivity of 504 mg/day.     

Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle.

Three experiments were conducted to examine effects of Buserelin, a potent agonist of gonadotropin-releasing hormone, on characteristics of ovarian follicles in cycling cows and heifers. In experiment 1, heifers were injected once with 10 micrograms Buserelin on Day 11, 12, or 13 of the estrous cycle (estrus = Day 0), or once with 20 micrograms of Buserelin on Day 12. Additionally, two groups were injected with a luteolytic dose of prostaglandin F2 alpha (PGF2 alpha) on Day 13 preceded with or without a Buserelin injection (10 micrograms) on Day 12. A control group did not receive a Buserelin injection. Ovaries were recovered and weighed after animals were slaughtered on Day 15. Follicle diameters were measured with calipers. Follicles for all experiments were classified as small (class 1: 3-5 mm diameter), medium (class 2: 6-9 mm), or large (class 3: greater than 9 mm). Heifers receiving only Buserelin had an increased number of medium-sized follicles compared to controls. Buserelin injection administered 24 h before PGF2 alpha reduced the decline in the average weight of the ovaries containing the corpus luteum (7.8 g for Buserelin before PGF2 alpha vs. 6.7 g for no Buserelin before PGF2 alpha). Buserelin pretreatment appeared to delay or prevent complete luteolysis by the injected PGF2 alpha. In experiment 2, 0, or 10 micrograms Buserelin was injected on Day 12 and follicle development was monitored by ultrasonography in situ from Day 12 to estrus. Follicles also were classified as clear or cloudy; cloudy was associated with flocculent material in the follicular fluid or with an indistinct follicular wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian responses to progesterone and oestradiol benzoate administered intravaginally during dioestrus in cattle

This study examined the effects of administering progesterone and oestradiol benzoate (ODB) during mid-dioestrus, on ovarian follicular dynamics in cattle. Twelve cycling cows were used in a 4 x 4 latin square design, with the 4 treatments being initiated on Day 13 of the cycle (oestrus = Day 0) and comprising intravaginal insertion for 5 days of: (i) a progesterone releasing device (CIDR; 'P4'); (ii) a CIDR device with a gelatin capsule containing 10 mg ODB and 1 g lactose (CIDIROL; 'P4/ODB') attached; (iii) a placebo CIDR device with the 10 mg ODB capsule (ODB); and, (iv) a placebo CIDR device alone (CTRL). The ovaries of each cow were examined daily by transrectal ultrasonography from Day 7 of the cycle until subsequent ovulation. Blood samples were collected daily from Day 11, and at intervals of 2-4 h during the 24 h period either side of treatment initiation. The second dominant follicle (DF2) emerged on Day 10.7 +/- 0.2 (mean +/- SEM), and was 8.5 +/- 0.2 mm in diameter by Day 13. The DF2 developed through to ovulation (2-wave cycles) in half of the animals in the CTRL group; while in the other half of cases, the ovulatory follicle originated from the third follicle wave that emerged on Day 17.2 +/- 0.4. Administration of a CIDR device alone (P4 group) did not alter the 1:1 ratio of 2 and 3-wave cycles, but the third dominant follicle (DF3) in those cows with 3-wave cycles emerged earlier on Day 15.6 +/- 0.2. In contrast, the DF2 of every animal in the ODB and P4/ODB groups became atretic and was replaced by a DF3 which emerged 4.0 +/- 0.3 days later. The effects of ODB on luteal function were limited to an earlier decline in plasma progesterone concentrations from 2 to 4 days after device insertion and a reduction in diameter of the corpus luteum when administered concurrently with progesterone. Intravaginal administration of 10 mg ODB on Day 13 of the oestrous cycle, with or without progesterone, was effective in promoting follicle wave turnover. In the absence of ODB, progesterone administration alone (P4 group) did not alter the ratio of animals with 2 or 3-wave cycles from that observed in animals in the CTRL group, but did advance the timing of subsequent follicle wave emergence in those animals with 3-wave cycles.     

Synchronisation of oestrus in dairy cows using prostaglandin F2alpha,gonadotrophin-releasing hormone,and oestradiol cypionate

An oestrous synchronisation protocol was developed for use in lactating dairy cows using PGF(2alpha), GnRH, and oestradiol cypionate (ECP). In experiment 1, lactating dairy cows received two injections of PGF(2alpha) (on days 0 and 11) (PP; n=10) or two injections of PGF(2alpha) (days 0 and 11) and 100 microg of GnRH on day 3 (PGP; n=10). In experiment 2, cows were treated with PGP (n=7), or PGP and 1 mg of ECP at the same time (PGPE(0); n=7) or 1 day after the second PGF(2alpha) injection (PGPE(1); n=7). In experiment 3, 101 lactating dairy cows in a commercial herd were assigned to one of three treatments; PP, PGP, or PGPE(1). Follicular growth was measured by ultrasound in experiments 1 and 2. Every cow (experiments 1, 2, and 3) was blood sampled at selected intervals for progesterone and oestradiol assays and inseminated at oestrus. In experiment 1, a higher percentage of GnRH-treated cows ovulated after the first PGF(2alpha) injection (90% versus 50%; P<0.05). The GnRH-treated cows tended to have a larger dominant follicle present at the time of the second PGF(2alpha) injection (16.5+/-0.5 mm versus 15.0+/-0.7 mm; P<0.10). The percentage of cows that ovulated after the second PGF(2alpha) injection was similar (60%). In experiment 2, cows treated with ECP had higher peak preovulatory concentrations of oestradiol in plasma (6.99+/-0.63 versus 3.63+/-0.63; P<0.01) following the second PGF(2alpha) injection and a higher percentage ovulated (86% versus 43%; P<0.05). A higher percentage of PGPE(1)-treated cows in experiment 3 were observed in standing oestrus and ovulated after the second PGF(2alpha) injection (standing oestrus, 26.4, 34.3, and 62.6%, P<0.01; ovulated, 56, 63, and 78%, P<0.05; PP, PGP, and PGPE(1), respectively). In conclusion, the PGP protocol increased the number of cows that ovulated after the first PGF(2alpha) injection and produced a more mature dominant follicle at the time of the second PGF(2alpha) injection. Adding ECP to PGP (PGPE(1)) enhanced the expression of oestrus and increased ovulation percentage. The combination of PGP and ECP is potentially a new method to routinely synchronise oestrus and ovulation in dairy cows.     

Plasma concentrations of inhibin a and follicle-stimulating hormone differ between cows with two or three waves of ovarian follicular development in a single estrous cycle

Patterns of ovarian follicle development were monitored daily in Holstein-Friesian cows that had two (n = 4) or three (n = 4) waves of ovarian follicle development during a single estrous cycle. The plasma from daily blood samples was used in assays for inhibin A, FSH, progesterone, and estradiol-17beta. Mean cycle lengths for cows with two and three waves were 21.8 and 25.3 days, respectively (P < 0.02). Although the average number of follicles >3-mm diameter on each pair of ovaries was similar for two- and three-wave cows on Days 2, 3, and 4 (Day 0 = day of ovulation; 8.6 vs. 9.6 follicles), there were more follicles >6-mm diameter on the ovaries of cows with two waves on Days 3 and 4. This difference was associated with a shorter interval from wave emergence to peak concentrations of inhibin A during the first wave in two-wave cows (2.0 vs. 3.8 days; P = 0.03) and with higher peak concentrations (474 vs. 332 pg/ml; P = 0.03). Differences in peak FSH concentrations were not significant (1.7 vs. 1.3 ng/ml; P = 0.10) and were inversely related to inhibin A concentrations. The peak concentrations of inhibin A and FSH in the second nonovulatory wave in the three-wave cows were similar to the low concentrations measured in the first wave (292 vs. 332 pg/ml of inhibin A, 1.3 vs. 1.3 ng/ml of FSH; P > 0.20). Average peak concentrations of inhibin A and FSH were similar during the ovulatory wave for cows with either two or three waves in a cycle (432 vs. 464 pg/ml of inhibin A, 2.3 vs. 2.1 ng/ml of FSH; P > 0.3). The lower concentrations of FSH during the emergence of the first follicular wave in cows with three-wave cycles may have reduced the rate of development of some of the follicles and reduced the concentrations of inhibin A. This pattern of lower concentrations of FSH and inhibin A was repeated in the second nonovulatory wave but not in the ovulatory wave. Subtle differences in the concentrations of these two hormones may underlie the mechanism that influences the number of waves of ovarian follicle development that occur during the bovine estrous cycle.     

Gibberellin A58 and ent-6α,7α,12α-trihydroxykaur-16-en-19-oic acid from seeds of cucurbita maxima

The isolation of gibberellin A₅₈ and ent-6α,7α,12α-trihydroxykaurenoic acid from a cellulase-hydrolysed extract of endosperm ofCucurbita maxima is described. The two compounds are characterized by their MS,¹H NMR and ¹³C NMR.     

Excretion rate of progesterone in milk and faeces in lactating dairy cows with two levels of milk yield.

This study was conducted to measure the effect of the level of daily milk yield on the excretion rate of progesterone (P4) in milk and faeces in high-producing (HP) and low-producing (LP) lactating dairy cows. A GnRH-agonist was implanted to block endogenous production of P4. A CIDR device was inserted into the vagina and left in place for 11 days. The average and peak milk yields were greater in HP cows (P < 0.0001). Mean plasma concentrations of P4 were also similar in both groups (P = 0.44), even though the average mass of P4 delivered from a CIDR device was higher with HP cows (P = 0.02). Average milk P4 concentration was similar in both groups (P = 0.81), so that average daily excretion of P4 in the milk was greater with HP cows (P = 0.05). The concentrations (P = 0.83) and daily yields (P = 0.4) of total faecal progesterone metabolites were not affected by level of milk yield. These data show that the concentrations of plasma and milk P4, and the concentration and yield of P4 metabolites are not affected by the levels of daily milk yield.