Efficiency of estrous synchronization in tropical sheep by combining short-interval cloprostenol-based protocols and “male effect”

This study assessed the efficacy of a protocol combining short-interval cloprostenol-based protocols and “male effect” for estrous synchronization in hair sheep. In Experiment 1, 24 ewes were randomly assigned to three groups (n = 8) and treated with cloprostenol on Days 3, 5 and 7 after ovulation, respectively. Estradiol secretion during the follicular phase was similar among groups. Onset of estrus (P < 0.001) and the timing of maximum LH concentration (P < 0.01) were earlier in group D3 than in D5 and D7 groups. During the subsequent cycle, the number and size of corpora lutea were higher (P < 0.05) in ewes of the groups D3 (1.9 ± 0.3 and 115.1 ± 14.3 mm²) and D5 (1.8 ± 0.2 and 100.2 ± 11.2 mm²) than in group D7 (1.3 ± 0.2 and 75.6 ± 6.4 mm²) group. In Experiment 2, 24 ewes were treated with two cloprostenol injections (7 days apart). Twelve ewes were exposed to “male effect” previous to an isolation period (ME group), whereas the remaining ewes were controls without male exposure (CTR group). Male effect induced earlier preovulatory LH surge (P < 0.05) and ovulation (P < 0.001) than CTR group. In Experiment 3, the estrus was synchronized in 68 ewes. Nineteen of them (group FGA) were treated using intravaginal sponges impregnated with fluorogestone acetate for 12 days and inseminated at 55 h. Forty-nine females (group ME) were treated like ME group. Twenty-four (ME48 group) and 25 ewes (ME55 group) were inseminated at 48 and 55 h after treatment, respectively. The fertility rate was numerically higher in ME48 than ME55 and FGA groups (62.5, 44.0 and 47.4%, respectively). In conclusions, the combined use of short-interval cloprostenol treatment and “male effect” may be an adequate alternative for synchronizing estrus and applying artificial insemination in hair sheep throughout the entire year.     

Intravaginal sponges to synchronize estrus decrease sexual attractiveness in ewes

Vaginal secretions are an important source of chemical signals, which affect ewes' attractiveness. Moreover, alterations of vaginal flora reduce sexual attractiveness of estrous ewes. As intravaginal sponges containing progestagens (widely used for estrous synchronization) affect vaginal flora, our aims were to determine if estrous ewes pretreated with intravaginal sponges were less attractive than ewes displaying spontaneous estrus, and if the addition of antibiotic to the sponge mitigated the decreased sexual attractiveness. Seventy-two estrous ewes were used in experiment 1: in 36, estrus was synchronized with commercial intravaginal sponges (50 mg medroxyprogesterone acetate for 14 days, group MAP1), whereas the other 36 were given a PGF2α analogue 19 to 20 days earlier and displayed spontaneous estrus (group C1). In experiment 2, 72 ewes were treated with intravaginal sponges for 14 days; for 36 ewes, the sponges contained 0.02 mg oxytetracycline (group Ox), whereas there was no antibiotic in the sponges for the remaining 36 ewes (group MAP2). In both experiments, sexual attractiveness was determined in 12 groups of six estrous ewes (three MAP1 vs. three C1, and three MAP2 vs. three Ox for Experiments 1 and 2, respectively) located in a 4 × 4 m pen. Courting and mating time that each ram spent with each ewe was recorded. After 5 min, the ewe with which the ram spent more time (most attractive ewe, ranked one, scale one to six) was taken out from the pen. The procedure was repeated until the ram ranked all six ewes, and repeated in the 12 groups in both experiments. In experiment 1, C1 ewes were more attractive than MAP1 ewes (ranks: 2.9 ± 0.3 vs. 4.1 ± 0.3, mean ± SEM, respectively; P < 0.002). In experiment 2, sexual attractiveness of MAP2 and Ox ewes was similar (3.5 ± 0.3 vs. 3.4 ± 0.3, respectively). We concluded that the use of intravaginal sponges impregnated with medroxyprogesterone acetate negatively affected ewes' sexual attractiveness, but this decrease was not mitigated by inclusion of a local antibiotic.     

Corpus luteum function following single and double ovulation during estrous cycle in Sanjabi ewes

This study compared the effect of double and single ovulation on serum progesterone concentrations and luteal characteristics in Sanjabi ewes at different days of the estrous cycle. The estrous cycles of 197 Sanjabi ewes were synchronized by a 12-day treatment with intravaginal sponges (Chronogest^®). Estrus was detected in 144 ewes 27–39 h after sponge removal. Daily blood samples were taken every morning and analyzed for serum progesterone (P4). Ewes were then transported to a local abattoir, where nine ewes were slaughtered on each experimental day (days 1–16 after estrus) for ovary collection. The ovarian follicles were measured and categorized by size (very small <2 mm; small 2–3.5 mm; medium 3.5–5 mm; large >5 mm). On each slaughter day, the number of corpora lutea per ewe was classified as single and double ovulation. The results show that the effect of dominant follicles was less during the mid-luteal phase. Ovulation rate of right, left and both ovaries were (54.9%), (23.6%) and (21.5%), respectively. The incidence of double ovulations was 40.2%. In the case of ewes exhibiting double ovulation, 46.6% occurred unilateral (ewes exhibited both ovulations on the right ovary); whereas 53.4% occurred bilateral (ewes exhibited ovulations on the right and left ovaries). Unilateral double ovulation was not observed in the left ovary. The right ovary appeared to play a significantly greater role in ewes showing single and double ovulations than the left ovary (P < 0.05). Serum progesterone concentration showed minimum and maximum levels of 0.29 ± 0.15 and 5.51 ± 0.75 ng/ml on days 16 and 11 post-estrous, respectively (P < 0.001). The mean volume of individual corpus lutea in ewes with single ovulations was significantly higher than in ewes with double ovulations (P < 0.01). However, the total volume of corpus lutea in ewes with single ovulation was significantly lower than in ewes with double ovulations in some days of estrous cycle (P < 0.01). The serum progesterone concentration was significantly higher in double than single ovulating animals on days 1–16 of the estrous cycle (P < 0.001). These results indicated a relatively high incidence of double ovulation in ewes associated with increasing total luteal volume and high circulating concentrations of progesterone.     

Plasma luteinizing hormone concentrations in cows given repeated treatments or three different doses of gonadotropin releasing hormone

We hypothesized that: (i) repeated GnRH treatments would increase the magnitude and duration of the LH surge and would increase progesterone (P4) concentrations after ovulation; and (ii) the release of pituitary LH would be greater in response to larger doses of GnRH. In Experiment 1, ovary-intact cows were given an intravaginal P4 (1.9 g) insert (CIDR) for 10 d and 500 μg cloprostenol (PGF) at CIDR removal to synchronize estrus. On Days 7 or 8 after estrus, cows received two PGF treatments (12 h apart) and 100 μg GnRH at 36 (Control), 36 and 38 (GnRH38), or 36 and 40 h (GnRH40) after the first PGF. Mean plasma LH concentration (ng/mL) was greater (P < 0.05) in GnRH38 (8.8 ± 1.1) than in Control (5.1 ± 1.3), with that in GnRH40 (5.8 ± 1.3) being intermediate. Although the duration (h) of the LH surge was longer in GnRH40 (8.0 ± 0.4) than in either GnRH38 (P < 0.05; 7.0 ± 0.3) or Control (P < 0.09; 7.1 ± 0.4), mean postovulatory P4 (ng/mL) was greater (P < 0.01) in Control (4.2 ± 0.7) than in GnRH38 (2.9 ± 0.6) or GnRH40 (3.0 ± 0.7) cows. In Experiment 2, ovariectomized cows were given a CIDR for 10 d and 2 mg of estradiol cypionate im at CIDR insertion. Thirty-six hours after CIDR removal, cows received, 50, 100, or 250 μg of GnRH. Cows given 250 μg GnRH released more LH (9.4 ± 1.4 ng/mL) than those given 50 or 100 μg (6.1 ± 1.3 and 5.4 ± 1.4 ng/mL, respectively), and had an LH surge of longer duration than those given 50 μg (6.8 ± 0.4 vs. 5.1 ± 0.3 h). In summary, ovary-intact cows in the GnRH38 group had greater mean and peak LH concentrations, but subsequent plasma P4 concentrations were lower than in Control cows. Ovariectomized cows given 250 μg GnRH had a greater pituitary release of LH.     

The GnRH antagonist acyline prevented ovulation, but did not affect ovarian follicular development or gestational corpora lutea in the domestic cat

Two experiments were conducted to investigate the effects of the GnRH antagonist acyline (330 μg/kg, given sc) on ovarian follicular development and ovulation, as well as on pregnancy maintenance in domestic cats. In the first experiment, seven queens in proestrus (total of 24 proestrus periods), were randomly assigned to treatment with either acyline (ACY; n = 17) or a placebo (PLC; n = 7). All queens were mated with a fertile tomcat. In the ACY and PLC groups, cessation of estrus occurred (mean ± SEM) 7.0 ± 1.3 and 7.0 ± 1.7 d after treatment (P > 0.1), ovulation occurred in 2 of 17 and all seven estrus periods (P < 0.05), and pregnancy rates were 1 of 16 and 7 of 7 (P < 0.05), respectively. In the ACY and PLC groups, intervals from treatment to the onset of the ensuing proestrus were 18.4 ± 1.7 and 120 ± 17.2 d. In the second experiment, 14 pregnant queens were randomly allocated, according to their mating date, to treatment with acyline in early pregnancy (from 20 to 25 d, n = 3), mid pregnancy (from 26 to 45 d; n = 4), late pregnancy (> 45 d; n = 3), or injection of a placebo in early (n = 1), mid (n = 2), or late pregnancy (n = 1). Ultrasonographic assessments of the uterus were done every second day for 2 wk post treatment, and serum progesterone (P₄) concentrations were determined before treatment, and at 7 and 14 d after treatment. No pregnancies were prematurely terminated and post-treatment P₄ concentrations did not differ among treatment groups (P > 0.1). In conclusion, in the domestic cat, GnRH withdrawal by acyline prevented ovulation when given in early follicular phase (proestrus), but did not significantly affect luteal function during pregnancy.     

Effects of treatment with a prostaglandin analogue on developmental dynamics and functionality of induced corpora lutea in goats

The aim of this study was to compare morphological and functional features of spontaneous and induced corpora lutea (CLs) in goats. Fourteen adult and cycling Anglo Nubian goats (Argentina) were randomly allocated to two groups: Group N (n = 7) included goats with natural spontaneous oestrus and Group PG (n = 7) included does in which oestrus was synchronized by the administration of two i.m. cloprostenol doses, 10 days apart. In both groups, oestrous behaviour was checked twice daily (Day of oestrus = Day 0) and daily transrectal ultrasonographies were performed for evaluating CLs and follicles dynamics through the complete subsequent oestrous cycle; the luteal activity was determined directly, in terms of progesterone (P4) secretion, and indirectly, by assessing effects of CL on follicular dynamics. All goats exhibited oestrous behaviour and ovulation without differences in ovulation rate (N: 1.67 ± 0.2, PG: 2.0 ± 0.1). The total luteal tissue area showed linear growth from Day 4 to Day 15 of oestrous cycle in all goats, but the developmental dynamics differed between groups, treated goats had larger area (P < 0.01). Plasma P4 concentrations also increased from Day 0 to Day 15 in all the does; however, from Day 5 to Day 15, treated does had a lower concentrations than the untreated group (P < 0.001). There were differences in the development of follicular waves between groups; assessment of size-distribution showed that treated group had a higher number of small and larger follicles (P < 0.05). The largest follicles recorded in treated goats had a higher maximum diameter both at the first (PG: 7.6 ± 0.8 mm; N: 4.9 ± 0.7 mm, P < 0.05) and second follicular waves (PG: 6.3 ± 1.4 mm; N: 5.0 ± 0.4 mm, P < 0.05) and a longer growth phase during the second wave (PG: 6.5 ± 1.7 days; N: 4.6 ± 0.7 days, P < 0.05), coincident with the period of maximal luteal secretion. In conclusion, synchronization of oestrus and ovulation by the administration of a prostaglandin analogue causes differences in developmental dynamics and functionality of induced corpora lutea when compared to natural spontaneous ovulation.     

The effect of porcine luteinizing hormone in the synchronization of ovulation and corpus luteum development in nonlactating cows

The objective of this study was to determine the effects of different doses of porcine luteinizing hormone (pLH) versus 100 μg gonadotropin-releasing hormone (GnRH) on ovulatory response (during diestrus and proestrus) and corpus luteum (CL) development in nonlactating cows. In Experiment 1, 75 cows received an intravaginal insert containing 1.9 g progesterone (P4) for 10 d to synchronize estrus (Day 0), with prostaglandin F_2α (PGF) at insert removal. On Day 5, all follicles ≥8 mm were ablated, and on Day 12, cows received 8, 12.5, or 25 mg pLH or 100 μg GnRH. Mean (±SEM) plasma P4 concentrations on Day 12 did not differ among treatments (5.6 ± 0.2 ng/mL). Mean plasma LH concentration was greatest (P < 0.01) in cows given 25 mg pLH (4.3 ± 0.4 ng/mL). The ovulatory response to 25 mg pLH (84%) or 100 μg GnRH (72%) was greater (P < 0.05) than that to 8 mg pLH (32%), but not different from that of 12.5 mg pLH (58%). In Experiment 2, 68 cows were given two injections of PGF 10 d apart to synchronize estrus (Day 0). On Day 7, cows received PGF, and, 36 h later, pLH or GnRH (as in Experiment 1). The interval from treatment to ovulation was most variable in cows given 8 mg pLH; only 65% of these cows ovulated during the initial 27 h versus 88% of cows given 25 mg pLH (P < 0.05). Cows given 25 mg pLH or 100 μg GnRH had larger CL area and greater plasma P4 concentrations (P < 0.05) than that of those given 8 mg pLH. In summary, diestrous cows given 25 mg pLH had the greatest plasma luteinizing hormone concentrations, but ovulatory response did not differ from that of those given 100 μg GnRH. Proestrous cows given 25 mg pLH or 100 μg GnRH had greater CL area and P4 concentrations than that of those given 8 mg pLH.     

Comparison of the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early vernal transition

The objective was to compare the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early spring transition. Starting January 30th, mares kept under ambient light were examined by transrectal ultrasonography. When a follicle ≥25 mm was detected, mares were assigned to one of two treatment groups, using a sequential alternating treatment design. In the eFSH group, mares (n = 18) were treated twice daily with eFSH (12.5 mg im) until they achieved a follicle ≥35 mm; hCG was given 36 h later. In the deslorelin group, mares (n = 18) were treated twice daily with deslorelin (63 μg im) until a follicle ≥35 mm was detected, and then they were given hCG. Estrous mares were inseminated with fresh semen. Eight days after ovulation, embryo recovery attempts were performed. In each group, 14/18 (78%) mares ovulated following the eFSH or deslorelin treatment regimes. The mean (95% CI) interval from treatment initiation to ovulation was 8.2 d (7.3, 8.9) and 7.2 d (6.2, 8.1) in the eFSH and deslorelin groups, respectively. In the eFSH group, the number of ovulations was significantly higher (mean ± S.E.M.; 3.4 ± 0.4 vs. 1.1 ± 0.1 ovulations), and more embryos were recovered (2.6 ± 0.5 vs. 0.4 ± 0.2 embryos/recovery attempt). We concluded that eFSH and deslorelin treatment regimes were equally effective in inducing ovulation in early transitional mares, within a predictable time of treatment; however, the eFSH regime increased the number of ovulations and embryos recovered per mare.     

Comparison of control of oestrus and ovulation in sheep by an ear implant (SC-21009) or by intravaginal sponge (cronolone or map)

Oestrus and ovulation were observed in 234 Galway ewes in the breeding season in a preliminary evaluation of an implant progestagen treatment. A miniature ear implant (3 mg SC-21009) was used in a comparison with two intravaginal sponges (30 mg Cronolone and 60 mg medroxy progesterone acetate (MAP)). Each progestagen treatment was used in conjunction with pregnant mare serum gonadotrophin (PMSG) (0, 375 i.u. and 750 i.u.). The percentage of ewes showing oestrus after treatment was significantly affected by progestagen (Cronolone, 95%; MAP, 71%; SC-21009, 74%; P < 0.01), but not by PMSG dose level (0.1 < P < 0.2). Oestrus onset was similar among treatments, but heats ended significantly earlier in implant sheep. Significantly (P < 0.01) more ewes ovulated following Cronolone treatment (92%) than following MAP (74%) or SC-21009 (71%), and there was also a significant effect of PMSG (0, 73%; 375 i.u., 86%; 750 i.u., 93%; P < 0.01). PMSG dose level had a highly significant effect on the mean ovulation rate (0, 1.23; 375 i.u., 1.52; 750 i.u., 2.12; P < 0.001).     

Ovarian and endocrine responses in tropical sheep treated with reduced doses of cloprostenol

The present study aimed to assess the efficacy of reduced doses of cloprostenol for synchronizing estrus and ovulation in hair sheep. With the aim to evaluate the luteolytic activity of reduced cloprostenol doses, a first experiment was performed using a relatively large (group H: 126 μg; n = 8), medium (group M: 68.25 μg; n = 6) and small (group L: 38.5 μg; n = 6) cloprostenol dose. Luteolysis was assessed at Days 3 and 6 after injection (Day 0) by progesterone concentrations (P₄) and transrectal ultrasonography (US). In Experiment 2, sheep were randomly assigned to the same three doses to evaluate a protocol for estrous synchronization using two injections administered 9 days apart. A third trial was performed with ewes treated (9 days apart) with the large dose (H = 126 μg; n = 12) and with a small dose adjusted for facilitating volume management (LA = 43.75 μg; n = 12). Presence of estrous cycling was determined in all the ewes by US and P₄ assay, at Days −9, −6, −2, 0 (Day of second cloprostenol injection), 8 and 11. Bleeding and US were done every 4 h from 16 h of the beginning of the estrus during the third trial to assess the preovulatory LH surge and timing of ovulation. Additionally, blood samples were drawn at Days 0, 1, 2 and 3 to assess estradiol (Experiments 2 and 3) and P₄ (Experiment 2) concentrations during the ovarian follicular phase. In all experiments, percentage of animals showing luteolysis, preovulatory follicular dynamics and function and percentage of ewes showing behavioral estrus in response to treatment was similar among groups. Timing of estrus for group H was earlier than group L (28.6 ± 1.8 h compared with 37.1 ± 2.4 h; P < 0.05). In the third trial, the preovulatory LH peak was higher in the LA group than group H, in terms of maximum mean concentration during the surge (27.7 ± 1.8 ng/mL compared with 21.3 ± 2.2 ng/mL; P < 0.05) and area under the curve (AUC; 183.4 ± 12.7 ng/mL compared with 127.7 ± 10.9 ng/mL; P < 0.01). However, timing of ovulation was similar for H and LA groups. Thereafter, ovulation rate and luteal function at Day 11 were similar. Current results demonstrate that reduced doses of cloprostenol may be applied in a practical manner for reproductive management of sheep, with the additional advantage of reducing treatment costs.     

Does injection of prostaglandin F(2alpha) (PGF2alpha) cause ovulation in anestrous Western White Face ewes?

In a previous study in our laboratory, treatment of non-prolific Western White Face (WWF) ewes with PGF(2 alpha) and intravaginal sponges containing medroxyprogesterone acetate (MAP) on approximately Day 8 of a cycle (Day 0 = first ovulation of the interovulatory interval) resulted in ovulations during the subsequent 6 days when MAP sponges were in place. Two experiments were performed on WWF ewes during anestrus to allow us to independently examine if such ovulations were due to the direct effects of PGF(2 alpha) on the ovary or to the effects of a rapid decrease in serum concentrations of progesterone at PGF(2 alpha)-induced luteolysis. Experiment 1: ewes fitted with MAP sponges for 6 days (n = 12) were injected with PGF(2 alpha) (n = 6; 15 mg im), or saline (n = 6) on the day of sponge insertion. Experiment 2: ewes received progesterone-releasing subcutaneous implants (n = 6) or empty implants (n = 5) for 5 days. Six hours prior to implant removal, all ewes received a MAP sponge, which remained in place for 6 days. Ewes from both experiments underwent ovarian ultrasonography and blood sampling once daily for 6 days before and twice daily for 6 days after sponge insertion. Additional blood samples were collected every 4 h during sponge treatment. Experiment 1: 4-6 (67%) PGF(2 alpha)-treated ewes ovulated approximately 1.5 days after PGF(2 alpha) injection; these ovulations were not preceded by estrus or a preovulatory surge release of LH, and resulted in transient corpora hemorrhagica (CH). The growth phase was longer (P < 0.05) and the growth rate slower (P < 0.05) in ovulating versus non-ovulating follicles in PGF(2 alpha)-treated ewes. Experiment 2: in ewes given progesterone implants, serum progesterone concentrations reached a peak (1.7 2 ng/mL; P < 0.001) on the day of implant removal and decreased to basal concentrations (<0.17 ng/mL; P < 0.001) within 24 h of implant removal. No ovulations occurred in either the treated or the control ewes. We concluded that ovulations occurring after PGF(2 alpha) injection, in the presence of a MAP sponge, could be due to a direct effect of PGF(2 alpha) at the ovarian level, rather than a sudden decline in circulating progesterone concentrations.     

Regulation of follicular activity and ovulation in ewes by exogenous progestagen

The success of estrus synchronization programs using progestagen sponges, particularly for fixed-time AI, varies considerably. In view of the recent evidence in cattle that exogenous progestins alter follicular dynamics, it may be that the stage of the estrous cycle at which the synchronization protocol is begun affects the synchrony of ovulation. The goal of this study was to evaluate the effect of medroxyprogesterone acetate (MAP) intravaginal sponges on follicular dynamics, luteal function and interval to ovulation when inserted at 3 stages of the estrous cycle. Sponges were inserted for 12 d beginning on either Day 0, 6 or 12 (n = 5) following ovulation. Ovarian activity was monitored using real-time ultrasound imaging during the treatment and the post-treatment estrous cycles. Information from the post-treatment cycle was used as a baseline to compare with the treatment cycle. Most ewes (79%) in the post-treatment cycle exhibited 3 follicular waves in an estrous cycle of 16 d, with the second wave follicles having smaller diameter (P < 0.001). Treatment with MAP increased the number of follicular waves from 3 to 4 or 5 when sponges were inserted on Days 6 and 12, respectively. Size of the largest follicle was smaller (P > 0.01) in waves in the early and middle of the 12-d MAP treatment period when compared with the last 4 days. This effect was most pronounced when endogenous progesterone concentrations were elevated concurrently with the presence of the sponge. Persistence of the ovulatory follicle was increased (P < 0.001) when sponges were inserted on Day 12, the only treatment where these follicles were under the influence of MAP in the absence of functional corpora lutea. Follicles were regressing at sponge removal in the Day 6 treatment, which resulted in a delay in emergence of ovulatory follicles, the LH surge and ovulation (P < 0.08) in relation to Day 0 and Day 12. Treatment with MAP sponges does not adequately synchronize estrus and ovulation among cyclic ewes due to the different follicular patterns that result depending on the stage of cycle at the time of sponge insertion.     

AluI polymorphism of the bovine growth hormone (GH) gene, resumption of ovarian cyclicity, milk production and loss of body condition at the onset of lactation in dairy cows

Relationships among GH genotype (AluI polymorphism), parity, metritis and interval from calving to first ovulation, milk production and body condition score (BCS) loss were determined in dairy cows (n = 307) on four large-scale farms in Hungary. Cows with systemic signs of puerperal metritis or mastitis were excluded. Time of the first postpartum (PP) ovulation was obtained from milk progesterone profiles. Based on GH genotype determination, groups of leucine homozygous cows (n = 246) and valine allele carriers (n = 61) were formed. All animals became cyclic during the study period. The average interval to first ovulation was 27.6 ± 0.69-d PP (mean ± S.D.). Genotype had no effect on the commencement of ovarian cyclicity. First ovulation occurred sooner after calving in pluriparous than in primiparous cows. The greater BCS loss cows had during the first 30-d PP, the longer they took to resume cyclic ovarian function. The interval from calving to first ovulation was substantially affected by farm, but not by mild cases of puerperal metritis. Genotype was not related to cumulative 30-d milk yield or BCS loss after calving. Primiparous cows had lower milk yield than pluriparous ones. Cows with metritis lost more body condition than healthy individuals in the first month postpartum. We concluded that, under field conditions, AluI polymorphism of the bovine GH gene had no effect on the interval from calving to first ovulation and could not be directly related to differences in milk yield and to the extent of BCS loss during the first month after calving in Holstein-Friesian cows.     

Amino acid analysis of spider dragline silk using H NMR

The amino acid composition of Nephila clavipes dragline silk fiber was determined by conducting ¹H nuclear magnetic resonance (NMR) spectroscopy experiments on acid-hydrolyzed material. N. clavipes dragline silk was found to consist of 43.0 ± 0.6% Gly, 29.3 ± 0.2% Ala, 9.1 ± 0.1% Glx, 4.0 ± 0.1% Leu, 3.3 ± 0.1% Tyr, 3.4 ± 0.2% Ser, 2.7 ± 0.1% Pro, 2.1 ± 0.1% Arg, 1.07 ± 0.05% Asx, 0.96 ± 0.05% Val, 0.48 ± 0.03% Thr, 0.35 ± 0.03% Phe, and 0.28 ± 0.03% Ile. Compared with standard chromatography-based amino acid analysis (AAA), the chemical resolution of NMR allows for an amino acid solution to be characterized without separation and is shown to provide considerably higher precision. This allows for more accurate statistics on the variability of amino acids in spider dragline silk. In general, this ¹H NMR AAA technique is applicable to a large range of proteins and peptides for precise composition characterization, especially when the precise content of a minor component is critical and relatively large amounts of sample are available (microgram to milligram quantities).     

Magnetic resonance microscopy of collagen mineralization

A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T₂) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T₂ values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T₂ values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 ± 0.02 for control strips to a maximum value of 0.31 ± 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils.     

Interruption of the canine estrous cycle with a low and a high dose of the GnRH antagonist, acyline

To test the efficacy and clinical safety of a low and high dose of the GnRH antagonist, acyline, on estrous cycle interruption and anovulation in female dogs, 20 proestrous (<3 d) bitches were randomly assigned to one of the following pharmacological protocols (given sc): acyline 110 μg/kg (ACY-L; n = 6); acyline 330 μg/kg (ACY-H; n = 8); or placebo (PLACE, n = 6). The animals were monitored (clinical and vaginal cytology examinations) daily for 60 d. Blood samples for serum progesterone serum concentrations were collected 14 d after treatment to determine if ovulation had occurred. Appearance of side effects and days to the onset of the first spontaneous estrous cycle after treatment were also recorded. In both ACY groups, but not the PLACE group, estrous cycles were interrupted after treatment (P < 0.05). The interval from treatment to estrus interruption in ACY-L and ACY-H groups was 3.0 ± 0.6 and 3.2 ± 0.2 d, respectively (LSM ± SEM; P > 0.05). In the PLACE bitches, physical, behavioral and cytological proestrus slowly progressed to estrus and diestrus. Ovulation was absent in all ACY, but not in PLACE bitches (P < 0.05). None of the females manifested side effects related to the treatments (P > 0.05). Spontaneous return to a normal estrous cycle during the study period occurred in all ACY (ACY-L 19.5 ± 2.7 d vs ACY-H 24.8 ± 2.0 d; P > 0.05), but in none of the PLACE bitches (P < 0.05). In conclusion, acyline efficiently, safely and reversibly interrupted an early phase of the estrous cycle in bitches by preventing ovulation.     

Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows

Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TAI) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2 × 2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5 ± 2.3 d postpartum, received a 3 mg norgestomet ear implant sc, plus 3 mg norgestomet and 5 mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54 h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100 μg gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means ± SEM, 1.53 ± 0.1 vs. 0.48 ± 0.1 mm/d; P < 0.0001), its diameter on Day 11 (11.4 ± 0.6 vs. 9.3 ± 0.7 mm; P = 0.03), as well as ovulation rate (80.8% vs. 50.0%, P = 0.02), whereas GnRH improved the synchrony of ovulation (72.0 ± 1.1 vs. 71.1 ± 2.0 h). In Experiment 2 (n = 599 cows, 40 to 120 d postpartum), pregnancy rates differed (P = 0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control, GnRH, eCG, and eCG + GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 33.8%, P = 0.002; and 48.0% vs 37.6%, P = 0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol.     

Effects of PMSG on progesterone levels in ewes treated with fluorogestone acetate or prostaglandin F2α

Twenty-four crossbred ewes were observed for oestrus during January. Blood samples, taken on days 0, 1, 2, 5, 9, 13, 15 and 17 of the oestrous cycle, were assayed for progesterone to establish a pretreatment (control) progesterone profile. In the middle of February, 12 ewes were treated with 40 mg fluorogestone acetate (FGA) impregnated intravaginal sponges for 12 days. At the time of sponge withdrawal, six ewes were treated with an intramuscular (IM) injection of 500 i.u. pregnant mares' serum gonadotrophin (PMSG). Twelve other ewes were treated with two injections of prostaglandin F_2α (PGF_2α) 15 mg each, 11 days apart; six ewes were treated with 500 i.u. PMSG IM at the time of the second PGF_2α injection. All ewes were observed for oestrus and blood samples, taken on days 0, 1, 2, 5, 9, 13, 15 and 17 of the first post-treatment oestrous cycle, were assayed for progesterone to establish a post-treatment progesterone profile. PMSG significantly increased the total progesterone in ewes treated with FGA but not in ewes treated with PGF_2α.     

Differential effects of nitrogen and phosphorus enrichment on growth of dwarf Avicennia marina mangroves

The effects of N and P enrichment were investigated on growth and physiological responses of dwarf Avicennia marina mangroves in a hypersaline (58 ± 8 psu) field site in Richards Bay, South Africa. It was hypothesized that at high salinities mangroves allocate more resources to roots than shoots, and that nutrient enrichment with N and P will shift resource allocation to shoots and enhance growth and productivity. In unvegetated areas of the dwarf zone, 1-year-old A. marina seedlings were planted in pots and enriched bimonthly with N, P, N + P, or remained unfertilized (control-C), and growth and morphology of plants were monitored for 2 years. Enrichment with N and N + P shifted resource allocation to shoots from 38% to 55%, and increased dry biomass accumulation by over 500%, compared to the control treatment. In the N and N + P treatments, plant height, number of leaves, leaf chlorophyll content and photosynthesis increased by over 50%, 330%, 30% and 30%, respectively, compared to the C and P treatments. Enrichment with N and N + P increased N concentrations in roots by over 60% (from 1.0 ± 0.1% to 1.6 ± 0.2% of dry mass) and in shoots by over 100% (from 1.3 ± 0.1% to 2.7 ± 02% of dry mass). Plants enriched with P alone were similar to those of the control. This study has demonstrated that dwarf A. marina in Richards Bay is N limited, and that N enrichment shifts resource allocation from roots to shoots and increases growth and productivity.     

A controlled internal drug-release dispenser containing progesterone for control of the estrous cycle of ewes

Experiments were conducted to evaluate a controlled internal drug-release (CIDR) dispenser containing progesterone to control the estrous cycle of ewes. After insertion of CIDR dispensers into the vaginae of ovariectomized ewes (Experiment 1; n = 11), the mean plasma progesterone rose from 0.74 ± 0.2 ng/ml to a peak of 5.5 ± 1.0 ng/ml within 2 h and then declined to 3.0 ± 0.5 ng/ml by 48 h. This was followed by a more gradual decline to 1.7 ± 0.3 ng/ml at the time of removal 12 or 14 d later. Following removal, the levels declined to baseline within 4 h. In Experiment 2, a 12- or 14-d treatment with CIDR dispensers was initiated in ewes 2, 9 and 16 d after synchronization of the estrous cycle with fluorogestone acetate (FGA)-impregnated intravaginal sponges. An intramuscular (i.m.) injection of 500 IU pregnant mare serum gonadotropin (PMSG) was given at the time of removal of the FGA sponge or CIDR dispenser. Based on plasma progesterone profiles, CIDR dispensers inserted 9 or 16 d after FGA sponge removal delayed the onset of a new estrous cycle until they were withdrawn. Following withdrawal, ovulation was effectively synchronized in all treatment groups and accompanied by development of functionally active corpora lutea with a normal lifespan. In Experiment 3, comparison of the mating response of ewes after treatment with CIDR dispensers (n = 192) or FGA sponges (n = 194) showed that 92% and 91% of the treated ewes, respectively, were marked by the ram within 72 h. Fertility and litter size of ewes bred at the synchronized and followup estrus were similar for both treatments. These results indicate that treatment of ewes with CIDR dispensers containing progesterone maintains plasma levels of progesterone within the range found during the normal estrous cycle. The CIDR dispenser is effective in synchronizing the estrous cycle of adult ewes and offers a promising alternative to the FGA-impregnated intravaginal sponge.