Biomedical subjects

J M Sreenan

Publications and source records attributed to J M Sreenan.

At least 19 recordsLinked to original sources

PubMed2002

Extent, pattern and factors associated with late embryonic loss in dairy cows.

Intensive genetic selection for increased milk production, coupled with increased dry matter intakes has led to significant improvements in cow milk yield, however, this increase in milk output has been accompanied by a decline in cow fertility. It has been suggested that there is a higher increment of late embryonic loss in high-yielding than in moderate yielding cows or in heifers. The objectives of this study were to establish the extent and pattern of embryonic loss, from days 28 to 84 of gestation, and to examine possible relationships between cow milk yield, cow genetic merit, parity, calving to insemination interval and embryonic loss in dairy cows managed mainly under pasture-based milk production systems. Multiparous dairy cows (n=1046) located on 8 farms and nulliparous dairy heifers (n=162) located on five of these farms were used in this study. The extent and timing of embryonic loss was measured by ultrasound scanning of the cows and heifers at 14-day intervals between days 28 and 84 of gestation. Positive diagnosis of pregnancy was based on the presence of an embryo or foetus with a visible heartbeat and, at the later scans, visible movement, whose size was compatible with stage of gestation and also on the presence of clear amniotic fluid of the cows and heifers presented as presumed pregnant on day 28 after insemination, 67 and 81%, respectively had a viable embryo. The subsequent embryonic loss rate between days 28 and 84 of gestation was similar (P>0.05) for cows (7.2%) and heifers (6.1%) and the pattern of loss over this period was also similar (P>0.05) for cows and heifers. There was no significant association (P>0.05) between level of milk production or milk energy output measured to day 120 of lactation and embryonic loss rate. Similarly, there was no significant relationship (P>0.05) between % milk fat, % milk protein and % milk lactose and embryonic loss rate. The extent and pattern of embryonic loss were not related (P>0.05) to either cow or to cow sire genetic merit. There was no significant (P>0.05) relationship between the calving to first service interval and embryonic loss. The extent of embryonic loss was greater (P<0.05) in cows that lost body condition between days 28 and 56 of gestation compared with cows than either maintained or improved in body condition.

Abortion, Veterinary↗

PubMed2002

Effect of elevated systemic concentrations of ammonia and urea on the metabolite and ionic composition of oviductal fluid in cattle.

High dietary protein leads to elevated systemic concentrations of ammonia and urea, and these, in turn, have been associated with reduced fertility in cattle. The effect of elevating systemic concentrations of ammonia and urea on the concentrations of electrolytes and nonelectrolytes in bovine oviductal fluid were studied using estrus-synchronized, nulliparous heifers (n = 25). Heifers were randomly assigned to 1 of 3 treatments consisting of jugular vein infusion with either ammonium chloride (n = 8), urea (n = 8), or saline (n = 9). Oviducts were catheterized, and fluid was recovered over a 3-h period on either Day 2 or 8 of the estrous cycle. No difference (P > 0.05) was found in the concentrations of any electrolyte or nonelectrolyte between oviducts ipsi- or contralateral to the corpus luteum. Plasma and oviductal concentrations of urea were increased by infusion with urea (P < 0.001) and ammonium chloride (P < 0.05) but not by saline (P > 0.05). Plasma and oviductal concentrations of ammonia were elevated by infusion with ammonium chloride (P < 0.001) but not by infusion with urea or saline (P > 0.05). No effect (P > 0.05) of treatment was found on oviductal or plasma concentrations of glucose, lactate, magnesium, potassium, or sodium or on plasma concentrations of insulin or progesterone. The concentration of calcium in oviductal fluid was reduced by urea infusion and was negatively associated with systemic and oviductal concentrations of urea. Oviductal concentrations of sodium were higher on Day 8 than on Day 2 (P < 0.05). No effect of sample day was found on any of the other electrolytes or nonelectrolytes measured (P > 0.05). Elevated systemic concentrations of ammonia and urea are unlikely to reduce embryo survival through disruptions in the oviductal environment.

Ammonia↗

PubMed2002

Amino acid turnover by elongating cattle blastocysts recovered on days 14-16 after insemination.

Blastocyst elongation from day 14 to day 16 after insemination coincides with a major phase of embryo loss in cattle. Protein synthesis, reflected in protein content, increases markedly over this period but little is known about the amino acid requirement of elongating blastocysts at this time. Cattle blastocysts produced in vivo were recovered on days 14-16 after insemination and cultured individually for up to 8 h in synthetic oviduct fluid containing a physiological mixture of amino acids plus 1 mmol glutamine l(-1) and 0.1% (w/v) polyvinyl alcohol (SOFaaPVA). After 1, 4 and 8 h in culture, an aliquot of culture medium was removed and the rate of amino acid depletion or production was calculated per unit of protein and per hour of culture. Amino acids were depleted or produced at different rates. Arginine was depleted from the medium at a significant rate (P < 0.05) during all culture periods. Alanine and glutamate were produced at a significant rate (P < 0.05) during all culture periods. The rate of alanine production was significantly greater (P < 0.05) in blastocysts recovered on day 14 compared with days 15 or 16 after insemination. Alanine production and arginine depletion tended to be greater in smaller embryos recovered on day 14 compared with larger and later stage embryos, indicating that earlier stage embryos may have higher metabolic activity than later stage embryos. Qualitatively, the pattern of amino acid consumption and production during elongation was similar to that shown from the zygote to early blastocyst stage.

Alanine↗

PubMed2000

The insulin-dependent glucose transporter isoform 4 is expressed in bovine blastocysts.

We have investigated the expression of two glucose transporter isoforms, Glut1 and 4, in 14- and 16-day-old bovine blastocysts (d14, d16) using RT-PCR, competitive RT-PCR and in situ hybridization. The blastocysts were grown in vivo or had been produced in vitro. Glut1 mRNA was detected in all blastocysts studied, Glut4 in all d14 blastocysts, but only in a few d16 blastocysts. Glut4 mRNA was localized in trophoblast and endoderm cells. Glut1 mRNA increased from d14 to d16 while Glut4 transcription was down-regulated in d16 blastocysts. The mRNA amounts varied between 0.8 to 23 pg and 3.9 to 65 fg per 100 ng embryonic RNA for Glut1 and Glut4, respectively, displaying a 100- to 1500-fold lower expression of Glut4 compared with Glut1 during blastocyst elongation. This is the first report on the expression of the insulin-sensitive Glut4 isoform in mammalian preimplantation embryos.

Animals↗

PubMed2000

The effect of dose and route of oestradiol benzoate administration on plasma concentrations of oestradiol and FSH in long-term ovariectomised heifers.

Oestradiol (E(2)) suppresses FSH and affects follicle wave dynamics in cattle. However, neither the optimum dose of ODB required to suppress FSH nor the effect of route of ODB administration on blood concentrations of E(2) are known; hence, the aim of this experiment was to answer these questions. Ovariectomised heifers received Progesterone Releasing Intravaginal Device (PRID) for 7 days, and 4 days later heifers received one of eight ODB treatments at second PRID insertion as follows; (1) 0.0 mg (Control; n=3), (2) 0.5 mg (n=4), (3) 1.0 mg (n=4), (4) 2.5 mg (n=6), (5) 5.0 mg (n=4), (6) 10. 0 mg (n=4), (7) 5.0 mg (n=4), and (8) 10.0 mg (n=5). For treatments 2-6 inclusive, ODB was administered intramuscularly in oil, while for treatments 7 and 8, the ODB in powder form was administered topically in the vagina by gelatine capsule attached to the PRID. Blood samples were collected every 6 h for the first 48 h, every 12 h for the next 48 h, and twice daily for a further 6 days. The interval from ODB administration to peak E(2) concentration was similar (P0.05) for treatments 2-6 where ODB was administered intramuscularly (mean 13.4+/-1.24 h), and was longer (P<0.05) for the intravaginal capsule treatments (mean 25.5+/-2.84 h). Plasma concentrations of E(2) increased with increasing intramuscular dose of ODB injected, (plasma E(2)=-0.237+16.109 (dose)-0.74 (dose)(2), R(2)=0.75; P<0.05). Peak plasma concentrations of E(2) following the 5- and 10-mg capsules were similar to each other and to those following the 0.5-mg injection (P0.05), but were lower than concentrations obtained following injection of 1.0-5.0 mg (P<0.05). Across all treatments, both the maximum percentage decline in FSH and the interval to FSH nadir were related to the peak plasma concentrations of E(2) (maximum % decline in FSH=11.17+1.564 (peak E(2))-0.009 (peak E(2))(2), R(2)=0.75; P<0.01), (hours to FSH nadir=10.628+1.486(hours to peak E(2))-0.0282(hours to peak E(2))(2), R(2)=0.22; P<0.05). Concentrations of FSH increased as E(2) declined from its peak value, irrespective of maximum value achieved. It was concluded that the intramuscular administration of ODB in oil to ovariectomised heifers given a PRID results in higher plasma concentrations of E(2) and causes a greater reduction in FSH than administration topically by intravaginal gelatine capsule. E(2) transiently suppresses FSH in ovariectomised heifers, and the magnitude of the suppression is dose-dependent; however FSH concentrations begin to increase 1-2 days after ODB administration while concentrations of E(2) were declining but still high.

Animals↗

PubMed2000

Embryo and foetal loss in beef heifers between day 14 of gestation and full term.

Following insemination, reproductive failure in cattle is largely manifested as embryo mortality and is a major source of financial loss to livestock producers. Ongoing studies at this laboratory into factors affecting embryo mortality have facilitated the collection of new data on the extent and timing of embryo and foetal mortality in cattle. Oestrus was synchronised in 158 beef cross heifers and following artificial insemination, embryo and foetal survival rates were determined on days 14 and 30 after insemination and subsequently at calving. Embryo survival rates measured on days 14, 30 and at full term were similar at 68%, 76% and 71.8%, respectively (P0.05). Based on morphological examination, all the 14-day-old embryos recovered were assessed as grade 1. These results provide new information indicating that most embryo losses in heifers have occurred before day 14 after insemination.

Abortion, Veterinary↗

PubMed2000

Uptake and incorporation of myo-inositol by bovine preimplantation embryos from two-cell to early blastocyst stages.

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo preimplantation cattle embryos was investigated using [(3)H] myo-inositol. Uptake of inositol was examined in two-cell and four-cell embryos (day 2 after insemination), morulae (day 6) and early blastocysts (day 7). Uptake in all stages examined was largely sodium-dependent indicating the presence of a sodium-dependent inositol transporter. Uptake of inositol did not vary significantly from two-cell to early blastocyst stages when expressed either on a per embryo or a per microg of protein basis. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP, and PtdInsP(2), was detectable at all stages examined. In contrast, incorporation of inositol into inositol phosphates was not detected until blastocyst formation at day 7. The second messenger, Ins(1,4,5)P(3), was first detected in day 7 blastocysts.

Animals↗

PubMed2000

Protein synthesis and phosphorylation by elongating 13-15-day-old cattle blastocysts.

Most early embryonic loss in cattle occurs during the period of blastocyst formation and elongation. In this study de novo protein synthesis and phosphorylation by 13-15-day-old cattle blastocysts was determined. Blastocysts were classified by dimension into spherical, ovoid and elongated stages and cultured under oil for 4 h in drops of KSOM medium containing either 35S-methionine or 32P-orthophosphate. Protein content increased with shape from spherical to ovoid to elongated. There were differences (P<0.05) within and among days in the percentage of radiolabel incorporated. Incorporation of 35S-methionine increased with embryo protein content. However, incorporation of 35S-methionine into TCA-insoluble material, expressed per microg of protein, decreased with shape (P<0.001) with more than a 10-fold difference between spherical and elongated 13-day-old blastocysts. Incorporation of 32P-orthophosphate also tended to decrease with shape, with a 3-fold difference between spherical and elongated Day 13 blastocysts. It was concluded that, from Day 13, protein synthesis and phosphorylation, when expressed per unit of protein, decreased from a much higher level, which in the present study was around the time the blastocysts began to elongate.

Animals↗

PubMed2000

The effect of acute nutritional change on follicle wave turnover, gonadotropin, and steroid concentration in beef heifers.

The effects of acute nutritional change on endocrine and ovarian characteristics were studied in cyclic (intact; n = 20) and long-term ovariectomized (ovx; n = 18) heifers being fed 1.2 x maintenance (1.2M). On d 7 of an 8-d progesterone and estradiol treatment, intact and ovx heifers were randomly allocated to diets providing .4, 1.2, or 2.0M until emergence of the second follicular wave after ovulation in intact heifers. In intact heifers, two of eight fed .4M failed to ovulate. In the other six, growth rate and maximum diameter (1.1+/-.09 mm/d and 10.1+/-.7 mm, respectively) of the first dominant follicle (DF) postovulation were less (P<.05) than in heifers fed either 1.2 (1.6+/-.18 mm/d; 12.9+/-.44 mm) or 2.0M (1.6+/-.08 mm/d; 12.7+/-.7 mm). In intact heifers, LH pulse frequency and amplitude were not affected by diet (P>.10). In ovx heifers, the frequency of LH pulses was unaffected by diet (P>.10), but heifers fed .4M had a greater pulse amplitude (P<.05) and mean concentration of LH (P<.001) than those fed 1.2 or 2.0M. Plasma concentrations of FSH were greater (P<.05) in ovx heifers fed .4M than in those fed 1.2 or 2.0M and increased linearly with time (P<.01). The FSH concentrations in heifers fed 1.2 and 2.0M were similar (P>.10) and decreased linearly with time (P<.001). In intact heifers, concentrations of FSH preceding follicle wave emergence were greater in heifers fed .4M (P<.001), but basal concentrations were not affected (P>.10). Concentrations of progesterone and estradiol were unaffected by diet (P>.10). Significant diet x ovarian status interactions in plasma IGF-I concentrations existed. Plasma concentrations of insulin increased as the level of nutrition increased, whereas concentrations of NEFA decreased. In conclusion, growth rate and maximum diameter of the DF were decreased by acute nutritional restriction, without affecting the concentration of LH. The magnitude of the FSH increase preceding new follicle wave emergence increased following dietary restriction, but concentrations of FSH were unaffected during the other stages of DF growth. The results of this study may have important implications for the feeding strategies adapted for high-yielding dairy cows in the early postpartum period when feed intake is often physiologically restricted.

Animal Nutritional Physiological Phenomena↗

PubMed2000

The effect of progesterone alone or in combination with estradiol on follicular dynamics, gonadotropin profiles, and estrus in beef cows following calf isolation and restricted suckling.

The effects of calf isolation and restricted suckling on LH pulse characteristics and interval to first ovulation (postpartum interval) were studied in 52 multiparous beef cows, with or without exogenous progesterone. At 30 d postpartum, cows were randomly allocated to one of four treatments (n = 13/treatment): 1) Ad lib, ad libitum access of cows to calves; 2) CI/RS, calf isolation/restricted suckling, where suckling was restricted to once daily; 3) CI/RS+P4, same as CI/RS but cows received an intravaginal progesterone-releasing device at calf isolation for 6 d; or 4) CI/RS+P4+E2, as CI/RS+P4 but the intravaginal progesterone-releasing device had a 10-mg estradiol capsule attached. Daily ovarian scanning and twice-daily blood sampling were performed from d 25 postpartum until the day of second ovulation. A random sample of cows from each treatment (n = 31 in total) were blood-sampled at 15-min intervals for 10 h on d 29, 32, 35, and 38. Ovulatory response to treatment was regarded as ovulation of either the dominant follicle growing at d 30 or the subsequent DF. There was a treatment x day effect (P = .09) on LH pulse frequency, but neither progesterone (CI/ RS+P4) nor progesterone and estradiol (CI/RS+P4+E2) treatment suppressed the calf isolation/restricted suckling-induced increase in LH pulse frequency. The estradiol capsule (CI/RS+P4+E2) delivered sufficient estradiol to delay new follicle wave emergence (treatment x stage; P < .001) and the associated preemergence increase in concentrations of FSH (treatment, P < .05) in cows treated at the postselection stage of follicle wave development, prolonging dominance of the dominant follicle present at treatment initiation (P < .001). The number of cows that ovulated in response to treatment was greater (P < .001) in cows with calf isolation/restricted suckling than in cows suckled ad libitum. Hence, cows assigned to the Ad lib treatment had a longer postpartum interval (P < .001) than cows of the other treatments. Exogenous progesterone treatment increased the frequency of cows exhibiting clinical signs of estrus at first ovulation (P < .001) and reduced the frequency of short estrous cycles (P < .001). We conclude that, in beef cows with calves, a 6-d progesterone treatment does not suppress the calf isolation/restricted suckling-induced increase in LH pulse frequency. Hence, on progesterone withdrawal, the LH pulse frequency is sufficient to stimulate first ovulation, accompanied by overt estrous expression and elimination of a short estrous cycle in most cows.

Animals↗

PubMed1999

Effect of acute nutritional restriction on incidence of anovulation and periovulatory estradiol and gonadotropin concentrations in beef heifers.

The effects of acute nutritional restriction on follicular dynamics, incidence of anovulation, and periovulatory estradiol and gonadotropin concentrations were studied in two replicates using beef heifers exhibiting regular estrous cycles. Heifers fed a diet supplying 1.2 maintenance (1.2 Mn) were synchronized using an intravaginal progesterone-releasing device for 8 days. One day before device removal, heifers were allocated randomly, within replicate, to a diet supplying 0.4 Mn (n = 20), or kept at 1.2 Mn (n = 21). On the sixth day after detected ovulation, heifers received 500 microg of synthetic prostaglandin F(2alpha) (PGF(2alpha)) to induce luteolysis, estrus, and ovulation of the first dominant follicle (DF). Animals were inseminated and returned to a diet of 1. 2 Mn. Pregnancy diagnosis was performed 30 days later. The maximum diameter subsequently attained by the DF present at progesterone withdrawal was smaller (P < 0.01) in heifers fed 0.4 Mn. Two heifers fed 0.4 Mn failed to ovulate this DF (P > 0.10). Growth rate (P < 0. 01) and maximum diameter (P < 0.001) of the DF in the first follicular wave of the next estrous cycle was also reduced in heifers fed 0.4 Mn. After prostaglandin administration, a further 10 heifers fed 0.4 Mn failed to ovulate the first DF of this cycle, and it regressed (P < 0.001), causing anovulation in 12 of 20 heifers within 13-15 days (P < 0.001). Anovulation of the DF present at progesterone withdrawal was preceded by a proestrous estradiol increase but absence of a gonadotropin surge (2 of 2 heifers), while neither endocrine event was detected before anovulation of the DF of the first new follicular wave (2 of 2 heifers). In cases in which ovulation of the first DF of the new cycle occurred, fertility was similar (P > 0.10) in heifers fed either 0.4 (n = 7) or 1.2 Mn (n = 20). In conclusion, acute nutritional restriction of cyclic heifers from 1.2 to 0.4 Mn decreased the growth rate and maximum diameter of DFs and induced failure of the DF to ovulate in 60% of heifers, but, within the confines of limited animal numbers, did not compromise fertility in heifers that ovulated.

Animal Nutritional Physiological Phenomena↗

PubMed1999

Immunization of rats and sheep against granulosa cell-inhibitory factor from bovine follicular fluid increases the number of large follicles in rats and the ovulation rate in sheep.

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.

Animals↗

PubMed1999

Effect of adenylyl cyclase activation on intracellular and extracellular cAMP and cGMP in preimplantation cattle blastocysts.

The effects of direct and indirect activation of adenylyl cyclase on the production of intracellular and extracellular cAMP and cGMP by 13- to 16-day-old cattle embryos were determined. Embryos were incubated for 2 h in a Krebs Ringer bicarbonate medium containing the phosphodiesterase inhibitor isobutyl-methylxanthine, to which stimulating agents forskolin (100 mumol l-1), cholera toxin (2 micrograms ml-1), or both were added. Total (intra- and extracellular) basal cAMP and cGMP concentrations ranged from 6.65 +/- 0.895 to 3.4 +/- 0.708 fmol microgram-1 protein in 13-day-old embryos and from 4.05 +/- 1.151 to 0.19 +/- 0.041 fmol microgram-1 protein in 16-day-old embryos. Forskolin induced an increase (P < 0.001) in cAMP that ranged from 5.4-fold on day 13 to 2.7-fold on day 16, whereas cholera toxin induced an increase (P < 0.001) that ranged from 30-fold at day 13 to 21-fold at day 16, similar to the effect of forskolin and cholera toxin combined. Individually, forskolin and cholera toxin had no effect on cGMP concentrations, but together they induced an increase (P < 0.05). cAMP (P < 0.01) and cGMP (P < 0.001) concentrations decreased with embryo age from day 13 to day 16 for all treatments; the decrease was greater for cGMP than cAMP (5-24-fold versus 1.6-3.3-fold, respectively). It is concluded that inducible adenylyl cyclase is present in 13- to 16-day-old cattle embryos and that the embryos secrete cAMP and cGMP into the incubation medium. In addition, basal and inducible concentrations of cAMP and cGMP decrease with embryo age from day 13 to day 16. These observations indicate that cAMP and cGMP may have a role in the rapid embryonic cell proliferation that occurs at this time or in signalling to the endometrium.

Adenylyl Cyclases↗

PubMed1998

Effect of calf isolation on follicular wave dynamics, gonadotropin and metabolic hormone changes, and interval to first ovulation in beef cows fed either of two energy levels postpartum.

The effects of postpartum energy intake, restricted suckling, and cow-calf isolation on concentrations of LH, FSH, growth hormone, and insulin-like growth factor-I (IGF-I) and on postpartum anestrous interval were determined by randomly allocating beef cows with a mean body condition score of 2.3 +/- 0.1 to receive either 80 MJ metabolizable energy (low-energy diet [L]; n = 51) or 120 MJ metabolizable energy (high-energy diet [H]; n = 52) per cow per day from calving. At 30 days postpartum, cows within diet were randomized to 1) have continued full access to their calves from birth to weaning (ad libitum suckling: ADLIB), 2) be suckled once-daily with their calves penned adjacent (restricted suckling, adjacent: RESADJ), 3) be isolated from all calves except for a once-daily suckling period (restricted suckling, isolated: RESISO). The mean postpartum interval was similar (p > 0.10) for L and H cows (62 and 63 days, respectively). RESADJ cows had a shorter (p < 0.05) postpartum interval than ADLIB cows, and RESISO cows had a shorter interval (p < 0.05) than RESADJ cows, with all effects independent (p > 0.10) of diet. FSH secretion pattern was not affected by diet, suckling treatment, sequential follicle wave number, or follicle wave retrospectively realigned to emergence of first ovulatory wave. Within 5 days of suckling restriction and calf isolation, the number of LH pulses increased from 0.18 to 0.48 pulses per hour (p < 0.05). Both mean LH and the mean number of LH pulses increased linearly (p < 0.01) during the six follicle waves up to the first ovulatory wave. From 80 days before, until the time of, first ovulation, growth hormone decreased (p < 0.05) while IGF-I increased (p < 0.05), irrespective of treatment. The results indicate that the "suckling effect" in beef cows is the major factor affecting the duration of the postpartum interval and suggests that the maternal bond is more important than suckling in regulating LH pulse frequency, the key endocrine factor determining whether or not a dominant follicles ovulates. Removal of the suckling effect resulted in a rapid increase in LH pulse frequency, which was not dependent on level of postpartum nutrition, at least within the nutritional limits of this study. Mean concentrations of FSH, unlike LH, did not vary with follicle wave number, suggesting that lack of FSH is not a major factor delaying the resumption of ovulation in postpartum beef cows.

Animals↗

PubMed1997

Effect of peptide to carrier ratio on the immune and ovarian response to inhibin immunization in cattle.

We report on the effects of the peptide to carrier ratio on the immune and biological response to inhibin immunization in cattle. A peptide sequence from the alpha C-subunit of bovine inhibin was synthesized and conjugated to human serum albumin (HSA) at ratios of 4.3 moles (L) and 13.1 moles (M) of peptide per mole of HSA. Hereford-cross heifers (n = 6 per group) were injected with 3 mg of one of the peptide conjugates at primary, followed by a booster injection (1.5 mg) 11 weeks later. Control heifers (n = 6) were injected with HSA only. Blood samples were taken at regular intervals to measure antibody titre. Ovulation rate was measured by ultrasonography. Antibodies were generated in both peptide immunized groups. Control heifers and group L heifers had 1 ovulation at all ovulatory cycles monitored. Ovulation rate was increased (P < 0.05) in group M immunized heifers, with four of six heifers having twin ovulations in the first cycle following boost. These data support those of previous studies which indicated that immunization against the alpha C-subunit of bovine inhibin significantly disrupted the mechanism(s) controlling ovulation rate in cattle. It also indicates that both the immune and associated biological response is dependent on the nature of the conjugate used for immunization, specifically the ratio of peptide to carrier.

Animals↗

PubMed1997

Cyclic AMP and cyclic GMP concentrations in, and efflux from, preimplantation cattle embryos.

Basal embryonic cAMP and cGMP concentrations and cAMP and cGMP accumulation in embryos and in the incubation medium were measured in cattle blastocysts recovered at days 14 (n = 23), 15 (n = 29) and 16 (n = 23) of pregnancy. Cyclic AMP and cGMP concentrations were measured by radioimmunoassay and the results expressed per microgram of protein, which was determined by the Pierce Micro BCA protein assay. Cyclic AMP and cGMP were present on each day. Basal embryonic cAMP was similar on days 14, 15, and 16, at 2.3., 2.5 and 2.6 fmol per microgram protein, respectively, while the concentration of cGMP was higher at day 14 (0.14 fmol per microgram protein than at either day 15 or 16 (0.06 and 0.05 fmol per microgram protein, respectively; P < 0.05), which were similar (P > 0.10). Basal embryonic cAMP concentrations were 15 to 60-fold higher than cGMP concentrations. Following a 2 h culture period in the presence or absence of the phosphodiesterase (PDE) inhibitor, isobutylmethylxanthine (IBMX), the accumulated concentrations of cAMP and cGMP were measured in the embryos and in the incubation medium. IBMX did not affect the concentrations of either cAMP or cGMP in the embryos but increased the concentrations of cAMP (P < 0.005) and cGMP (P < 0.01) in the incubation medium. There was no effect of day on either embryonic or medium cAMP, but both embryonic and medium cGMP were higher at day 14 than at day 15 (P < 0.05) or 16 (P < 0.005). Differences between embryonic and medium cAMP and cGMP accumulation were examined. There was no effect of day or treatment. cGMP accumulation in the medium was higher than in the embryos (P < 0.005) whereas cAMP accumulation in the medium was lower than in the embryos (P < 0.05). Whether the efflux of cAMP and cGMP is active or passive is not clear but it may indicate a possible role in embryo-maternal signalling.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Protein content of cattle oocytes and embryos from the two-cell to the elongated blastocyst stage at day 16.

The protein content of cattle oocytes and preimplantation embryos produced in vivo, from the two-cell to the elongated blastocyst at day 16, was determined. From the oocyte to the expanded blastocyst stage (day 8), protein determination was carried out on zona pellucida-enclosed embryos. Protein content was measured by the Pierce Micro BCA protein assay. The mean protein content of oocytes was 0.126 micrograms, with no significant increase at the two-cell stage (0.132 micrograms). Protein content was higher at the morula stage (0.183 micrograms; P < 0.05) with a further increase at the expanded blastocyst stage (0.367 micrograms; P < 0.05). There was a 160-fold increase in protein content from the expanded blastocyst to the hatched day 13 stage. Spherical, ovoid and elongated blastocysts were collected on days 13 and 14. The mean protein content of day 13 (59.8 micrograms) and day 14 (92.4 micrograms) embryos was similar (P > 0.1), but the protein content of the elongated embryos was higher than that of ovoid or spherical embryos collected on the same day. Protein content of day 15 embryos (362.2 micrograms) was higher than that on day 14, with a further increase to 946.6 micrograms by day 16. The correlation between protein content and day of development contained both a linear and a quadratic component. Embryo length and width increased from day 13 (5.24 mm and 0.89 mm, respectively) to day 16 (51.6 mm and 1.82 mm, respectively). From day 13 to day 16, the protein content was correlated with both embryo length and width (r2 = 0.89 and 0.51, respectively; P < 0.001) and was highly correlated (r2 = 0.95) with the product of embryo length by width, indicating that protein content increases as a function of surface area.

Animals↗

PubMed1996

Partial purification from bovine follicular fluid of a factor of low molecular mass with inhibitory effects on the proliferation of bovine granulosa cells in vitro and on rat follicular development in vivo.

Bovine follicular fluid was aspirated from follicles of 2-20 mm in diameter, charcoal-treated to remove steroids and then separated into low and high molecular mass fractions. The low molecular mass (< 10 kDa) fraction was purified on a Sephadex G-25 chromatography column with formic acid as the eluent. Seven peaks were isolated and assayed for biological activity in cultures of bovine granulosa cells at concentrations of 10, 100 and 1000 ng ml-1. One peak (peak 4) inhibited (P < 0.001) the proliferation of granulosa cells when measured by cell counting and by [3H]thymidine incorporation (33-37% inhibition). This peak inhibited proliferation of granulosa cells from both small (< 2 mm) and medium (2-10 mm) follicles, but not large (> 10 mm) follicles. The inhibitory effect of peak 4 was not due to a toxic effect on cells. Administration of peak 4 to rats did not affect liver or kidney masses but did decrease uterine (25%, P < 0.01) and ovarian (35%, P < 0.01) masses. Peak 4 also caused a reduction in the number of large follicles (65%, P < 0.01) but increased the number of small follicles (55%, P < 0.01). We have named the inhibitory factor associated with peak 4, granulosa cell-inhibitory factor (GCIF). The results presented suggest that GCIF may be a factor secreted by dominant follicles that inhibits the development of subordinate follicles.

Animals↗