Natural acidic deep eutectic solvent to obtain cellulose nanocrystals using the design of experience approach.

In this study, a new approach to optimize the cellulose nanocrystals (CNCs) extraction using acidic natural deep eutectic solvents (NADES) was introduced using, for the first time, design of experiment method. Choline chloride:oxalic acid dihydrate with a molar ratio of 1:1 was used to extract CNCs. Then, three most important parameters were varied to design the experiment: (i) cotton fibre concentrations, (ii) temperature and (iii) treatment time. Two outcomes were studied: the CNC yield and the crystallinity. The mathematical model for crystallinity perfectly described the experiments, while the model for CNC yield provided only a tendency. For a reaction time of 6 h at 95 °C with a fibre concentration of 2 %, the expected optimum CNC yield was approximately 35.5 ±â€¯2.7 % with a crystallinity index of 80 ±â€¯1 %. The obtained experimental results confirmed the models with 43.6 ±â€¯1.9 % and 81 ±â€¯1 % for the CNC yield and the crystallinity index, respectively. This study shows that it is possible to predict the CNC yield CNC and their crystallinity thanks to predictive mathematical models, which gives a great advantage to consider in the near future a scale up of the extraction of cellulose nanocrystals using this original family of green solvents.

Pharmacokinetics and local tolerance of cefovecin sodium after intra-articular administration in horses.

Searching for new therapeutic options against septic arthritis in horses, this research was focused on the study of the kinetics and local side effects after the intra-articular treatment of horses with cefovecin sodium. A single dose (240 mg) of the drug (Convenia® ) was administered into the radiocarpal joint of adult healthy horses (n = 6), and drug concentrations in plasma and synovial fluid were determined by high-performance liquid chromatography (HPLC). Local tolerance was also studied based on the modification of different joint physiopathological parameters (pH, cellular, and protein concentration in synovial fluid). Although no clinically relevant joint damage was noticed, significant increases in the protein concentrations at 5 min and in the cellular concentration at 30 min after cefovecin administration were observed in the treated radiocarpal joints. The duration of the cefovecin above the minimal inhibitory concentration (MIC) ≤1 µg/mL was 28.80 ± 2.58 h in the radiocarpal joint and 16.00 ± 2.86 h in plasma. The results of this study showed that intra-articular administration of cefovecin sodium in horses could be considered in the future to manage septic arthritis in horses, as it offers a good pharmacokinetic behavior and good local tolerance.

Pharmacokinetics of meloxicam after intravenous, intramuscular and oral administration of a single dose to African grey parrots (Psittacus erithacus).

Meloxicam is a nonsteroidal anti-inflammatory drug commonly used in avian species. In this study, the pharmacokinetic parameters for meloxicam were determined following single intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) administrations of the drug (1 mg/kg·b.w.) in adult African grey parrots (Psittacus erithacus; n = 6). Serial plasma samples were collected and meloxicam concentrations were determined using a validated high-performance liquid chromatography assay. A noncompartmental pharmacokinetic analysis was performed. No undesirable side effects were observed during the study. After i.v. administration, the volume of distribution, clearance and elimination half-life were 90.6 ± 4.1 mL/kg, 2.18 ± 0.25 mL/h/kg and 31.4 ± 4.6 h, respectively. The peak mean ± SD plasma concentration was 8.32 ± 0.95 µg/mL at 30 min after i.m. administration. Oral administration resulted in a slower absorption (tmax  = 13.2 ± 3.5 h; Cmax  = 4.69 ± 0.75 µg/mL) and a lower bioavailability (38.1 ± 3.6%) than for i.m. (78.4 ± 5.5%) route. At 24 h, concentrations were 5.90 ± 0.28 µg/mL for i.v., 4.59 ± 0.36 µg/mL for i.m. and 3.21 ± 0.34 µg/mL for p.o. administrations and were higher than those published for Hispaniolan Amazon parrots at 12 h with predicted analgesic effects.

Long-lasting concentrations of cefovecin after subcutaneous and intramuscular administration to Patagonian sea lions (Otaria flavescens).

Cefovecin is a third-generation cephalosporin developed as an aqueous solution for use by the subcutaneous route in dogs and cats. This study evaluated the duration of cefovecin plasma concentrations after single intramuscular (IM) or subcutaneous (SC) injection at different doses in 10 Patagonian sea lions (Otaria flavescens). Blood samples were collected serially from the day of the injection up to 60-90 days post-injection. Plasma drug concentrations were determined by high performance liquid chromatography-UV detection and pharmacokinetic parameters were calculated by non-compartmental analysis. No reactions or side effects associated with the drug were observed in any of the studied animals. Both routes showed very similar pharmacokinetic behaviour. Elimination half-life (11.3-21.6 days, SC; 13.1-15.9 days, IM) and mean residence time (17.6-36.8 days SC; 16.5-25.4 days IM) were, in all cases and doses, considerably longer than those previously reported for any other species. Based on these findings, and preliminary data on specific pathogen sensitivity, cefovecin was found to be a very promising antimicrobial for Patagonian sea lions, in particular those that are difficult to access or that are under certain rehabilitation conditions.

Prepuberal evolution of plasma leptin levels in gilts of thrifty genotype (Iberian pig) and lean commercial crosses (Large White × Landrace).

Current study aimed to determine possible differences in plasma leptin levels during the prepuberal period and their relationship with the onset of puberty in gilts of obese thrifty genotype (Iberian breed) and lean genotype (Large White × Landrace commercial crosses) reared under similar conditions. Plasma leptin concentration increased linearly during the 7 weeks prior to the day of puberty attainment in both genotypes (P<0.005, r=0.707 for LW × L and P<0.0005, r=0.874 for Iberian gilts). However, leptin levels in the Iberian gilts was higher from the first sample of the experimental period, with females having 16 weeks-old (2.7±0.3 vs 1.7±0.2 ng/ml in LW × L; P<0.001), to the onset of puberty (8.5±0.7 vs 2.8±0.3 ng/ml in LW × L; P<0.005). Thus, the current study reinforces previous data on changes in around puberty and evidences, for the first time, profound differences in prepuberal plasma leptin levels between gilts of obese (Iberian) and lean genotypes (LW×L).

Patterns of corpora lutea growth and progesterone secretion in sows with thrifty genotype and leptin resistance due to leptin receptor gene polymorphisms (Iberian pig).

The current study aimed to compare luteal function, as measured by corpora lutea dynamics and progesterone secretion, in 10 sows with obesity/leptin resistance genotype (Iberian pig) and 10 females of lean commercial crosses (Large White × Landrace). In all the animals, the oestrous cycle was synchronized with progestagens, and ovulation was induced by exogenous gonadotrophins. Thereafter, number and size of follicles and plasma oestradiol concentration were determined at oestrus detection, and number and size of corpora lutea and progesterone concentration were evaluated from Day 4 to 12 of the cycle. There were no differences between genotypes in follicle population and oestradiol concentration, and ovulation rate (15.2±1.3 in Iberian vs 12.7±1.8 in LWxL sows); however, there was a higher percentage of Iberian than control sows showing luteal cysts (66.7% vs 30%, respectively; p<0.05). In both breeds, both total luteal area and plasma progesterone concentration grew linearly from Day 4 to 8 (p<0.01) and remained more stable between Days 8 and 12, without significant differences between genotypes. In conclusion, current study supports that ovulatory processes and luteal functionality are not the main limiting factors for prolificacy in a pig model of leptin resistance and obesity.

Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors.

The Mediterranean Iberian pigs are obese genotypes, due to a leptin resistance syndrome related to leptin receptor gene polymorphisms. The Iberian pig is affected by a lower prolificacy when compared to lean breeds, and thus may constitute a good animal model for adverse effects of obesity in reproductive performance. The present study determined possible differences in rates of ovulation and embryo implantation and later incidence of embryo mortality and intrauterine growth retardation (IUGR) in sows of Iberian breed (n = 23) and highly-prolific lean commercial crosses (Large White × Landrace, n = 17) at two critical periods of swine pregnancy: Day 21 (just after achievement of trophoblast attachment) and Day 35 (just after completion of the transition from late embryo to early foetal stage). Two different reproductive performances were identified in the Iberian breed; 58.3% of the females had lower ovulation rates than LWxL (13.2 ± 2.3 vs 22.5 ± 1.6, P < 0.05), but 41.7% had a similar number of ovulations (18.2 ± 3.9). However, those Iberian sows having high ovulatory rates showed a high incidence of regression of corpora lutea and embryo losses between Days 21 and 35, which was not found in Iberian females with low ovulation rates; therefore, the number of viable embryos was similar in both Iberian groups (8.2 ± 1.0 and 8.4 ± 1.0) and lower that in highly-prolific LWxL (14.8 ± 1.8, P < 0.05). At Day 35, a total of 167 conceptuses (73 LWxL and 94 IB) were evaluated for IUGR. The LWxL conceptuses were longer and wider than Iberian (69.5 ± 0.1 and 64.4 ± 0.1 vs 49.9 ± 0.1 and 41.9 ± 0.1 mm, P < 0.0001). However, Iberian conceptuses were heavier than LWxL (107.4 ± 6.6 vs 68.6 ± 2.4 g) due to a lower quantity of fluids and a higher development of the placental tissues in comparison to the embryo itself. In conclusion, current study indicates a bimodal effect of obese genotypes on reproductive performances, either by lowering ovulation rate or by increased embryo losses in the first third of pregnancy.

Preovulatory follicle dynamics and ovulatory efficiency in sows with thrifty genotype and leptin resistance due to leptin receptor gene polymorphisms (Iberian pig).

This study compares follicular function and ovulatory efficiency in 20 sows with obesity/leptin resistance genotype (Iberian pig) and 20 females of lean commercial crosses (Large White × Landrace; LW×L). Estrous cycle was synchronized with progestagens; ovulation was induced with eCG and hCG, in half of the females of each group, to determine its effect. In females of both breeds not treated with gonadotropins, the number of follicles larger than 4.9 mm and the estradiol secretion increased throughout the follicular phase (P<0.05); estradiol values were similar at estrus detection (22.5±1.2 vs. 26.5±0.6 pg/ml respectively, for Iberian and LW×L sows). Moreover, ovulation rate was higher in Iberian pigs (15.3±1.3 CLs) than in LW×L (10.2±1.3 CLs; P<0.05), with mean progesterone values being 18.1±0.7 ng/ml in Iberian and 16.8±0.6 ng/ml in LW×L pigs. Thus, the preovulatory follicular growth and the ovulatory efficiency seem not to be the main limiting factors for reproductive efficiency in Iberian swine. The gonadotropins induced a significant increase, when compared to untreated females (P<0.05), in the number of follicles larger than 4.9 mm growing throughout the follicular phase; however, estradiol values at estrus were lower (P<0.05) in both breeds (9.2±0.7 pg/ml in Iberian vs. 8.6±0.8 pg/ml in LW×L), when compared with the nontreated animals, which suggests defective follicular function after gonadotropin stimulation. There were also no differences between genotypes in ovulation rate (15.2±1.3 vs. 12.7±1.8) and progesterone secretion (21.2±0.8 ng/ml in Iberian and 20.9±0.7 ng/ml in LW×L sows) in the treated animals. In conclusion, the current findings indicate that preovulatory follicular growth and ovulatory efficiency are not main limiting factors for prolificacy in a pig model of leptin resistance and obesity.

Pregnancy-associated changes in plasma concentration of the endocrine disruptor di(2-ethylhexyl) phthalate in a sheep model.

The plasticizer di(2-ethylhexyl)phthalate (DEHP), used for producing polyvinyl chloride (PVC), acts as an endocrine disruptor with toxic effects on reproductive and developmental processes. Exposure to DEHP in humans is mainly by environment and food. Thus, our aim was to determine plasma levels in livestock animals using the ewe (Ovis aries) as a model. In a first trial, 150 samples from ewes of different ages (2 to 7 yr) and reproductive status (pregnant and nonpregnant) were analyzed by high-performance liquid chromatography (HPLC). DEHP was detected in 34.7% of the samples, with a mean level of 0.45+/-0.01mug/mL (range, 0.05 to 2.81mug/mL). The percentage of nonpregnant animals with DEHP traces was higher in animals older than 4 yr (n=66, 37.9%) than in younger animals (n=69, 17.4%; P<0.05), although the mean levels in ewes with residues were similar (0.16+/-0.01 vs. 0.16+/-0.02mug/mL). All the pregnant ewes (n=15) showed presence of DEHP, with higher plasma levels than that in nonpregnant females (1.42+/-0.18 vs. 0.16+/-0.01mug/mL; P<0.0001). For confirming the effect of pregnancy on mobilization of DEHP from body fat, 101 ewes of the same age were sampled in a second trial at a different farm. The percentage of animals with DEHP traces was higher in pregnant ewes (n=32, 71.9%; P<0.005) than in nonpregnant ewes (n=37, 35.1%) or in ewes that recently gave birth (n=32, 21.9%), although mean levels were similar (0.42+/-0.02, 0.33+/-0.02, and 0.34+/-0.05mug/mL, respectively). In conclusion, current results indicate a high incidence of ewes reared in the field showing accumulation of phthalates; percentage of animals with presence of DEHP increases with age, due to an extended period of exposure, but mainly during pregnancy, due to the mobilization of body reserves.

Advanced onset of puberty in gilts of thrifty genotype (Iberian pig).

The Iberian pig is an autochthonous breed of the Mediterranean area, reared extensively in the central and southern areas of Spain and Portugal and that is known worldwide for the production of Iberian ham. The characteristics of the Iberian ham are related to its abundance of intramuscular fat, owing to the high capacity of the pig to accumulate fat under its skin and between the muscular fibres. This ability to store excess fat enables survival during periods of scarcity and it has been found in other antique animal breeds and even in humans, being named as the thrifty genotype. The reproductive management of the Iberian pig, in spite of a lack of accurate information unlike in other swine breeds, is based on the assumption of lower reproductive precocity and efficiency than the modern commercial breeds. The current study characterized and compared the onset of puberty in gilts of Iberian breed and meat commercial crosses (Large White × Landrace) reared in the same conditions by assessing weekly plasma progesterone profiles. At the end of the experimental period, when the gilts became 180 days old, the percentage of Iberian females that had reached puberty was 72.2%, with a mean age and weight of 160.5 ± 2.6 days and 92.9 ± 4.7 kg, respectively. The percentage of Large White × Landrace cross-breed females reaching puberty at 180 days was only 15% (p < 0.05). The mean age and weight were 165.0 ± 4.1 days and 107.8 ± 2.2 kg (p < 0.05), respectively. Thus, converse to the traditional assumption, onset of puberty was advanced in Iberian gilts. These results are important for the reproductive management of the Iberian pig but, at the same time, may set the basis for future studies on the metabolism-reproduction link and, specifically, on the possible relationship between earlier accumulation of fat and attainment of puberty.

Accuracy of in vivo and ex vivo ultrasonographic evaluation of ovarian follicles and corpora lutea in sows.

Current study determined, in sows, the accuracy of ultrasonography for in vivo (n=8) and ex vivo (n=7) evaluation of corpora lutea (CLs) and follicles > or =1.5mm in size, by comparison with macroscopic findings in sliced ovaries. The accuracy for ex vivo detection of follicles increased with follicle size (P<0.05), being low for 1.5-1.9 mm follicles (65.9%) and higher for > or =6mm follicles (93.3%); differences between ultrasonographic and macroscopic observations were significant only for follicles smaller than 3.9 mm (P<0.05), due to underestimation. Ex vivo observation succeeded to detect presence or absence of CLs in all the ovaries; the efficiency for determining the exact number of CLs being 94.4%. The accuracy for in vivo detection of follicles also increased with follicle size (P<0.05), dropping to values lower than 40% for 1.5-1.9 mm follicles; therefore, there were significant differences between ultrasonographic and macroscopic observations (P<0.05). On the other hand, accuracy remained around 92% for > or =6mm follicles. Ultrasonography was useful again for detecting presence of CLs in all the ovaries; the efficiency for determining CLs number reached 86.7%, due to underestimation in ovaries with higher number of CLs (P<0.05). Overall, there were no significant differences when comparing the accuracy of ex vivo and in vivo scannings for determination neither of the number of follicles in each size-category larger than 1.9 mm nor of the presence of ovulations or of the CLs number in each ovary. In conclusion, the use of ultrasonography allows an accurate detection of the presence and number of CLs and follicles > or =2mm of diameter in sows, without significant differences between in vivo and ex vivo observations.

Glucogenic supply increases ovulation rate by modifying follicle recruitment and subsequent development of preovulatory follicles without effects on ghrelin secretion.

This study determined the effects of short-term energy inputs on ghrelin secretion and possible links with changes in the follicle population or the ovulation rate. Oestrous cycle was synchronized in 16 Manchega sheep using progestagen sponges and cloprostenol. Half of the animals were treated from days 0 to 4 by the oral administration, twice daily, of 200 ml of a glucogenic mixture containing 70% of glycerol, 20% of 1,2-propanediol and 10% of water; the control group received 200 ml water. The mean (+/-S.E.M.) plasma glucose increased immediately after the first administration (3.9+/-0.3 vs 3.0+/-0.1 mmol/l in control group, P<0.05), remaining statistically different during the treatment. However, plasma ghrelin levels were similar in both groups. On the other hand, the results indicated that short-term energy inputs modify ovulation rate (1.9+/-0.1 vs 1.3+/-0.2 in control group, P<0.05) by increasing the number of follicles able to be selected to ovulate during the period of treatment (>or=4 mm in size; 5.9+/-0.6 vs 4.3+/-0.4 at day 2, P<0.05). After sponge withdrawal, the number of these follicles decreased throughout follicular phase (5.8+/-0.8 to 1.5+/-0.4, P<0.0005) while the number of large follicles increased (>or=6 mm in size; 0.8+/-0.4 to 2.0+/-0.3, P<0.05); this would indicate an active growth of preovulatory follicles that were not found in the control group. Thus, the increases of ovulation rate by high-energy inputs would be caused by an enhancement in the developmental competence of preovulatory follicles.

Contribution of fish consumption to heavy metals exposure in women of childbearing age from a Mediterranean country (Spain).

The concentrations of cadmium (Cd), lead (Pb) and mercury (Hg) were determined in samples from sword-fish (Xiphias gladius, n=37) and louvar (Luvarus imperialis, n=17) taken in an official surveillance programme in Madrid (Spain). The levels of heavy metals were measured by atomic absorption spectrophotometry (hydride generator for Hg and flame atomic absorption for Cd and Pb) after digestion of the samples (microwave digestion for Hg and drying chamber for Cd and Pb). Pb contamination was negligible in both species. Cd was detected in 89.19% of the samples from sword-fish and 52.94% from louver; 17.65% of louvar and none from sword-fish being higher than maximum residue limits (MRL). However, around 90% of louvar samples and around 35% of sword-fish portions exceeded MRL for Hg; around 30% of louvar samples having levels even higher than 1 microg/g. The consumption of one serving of 150 g either of louvar or sword-fish would not be relevant for Cd and Pb provisional tolerable weekly intake (PTWI) of these elements for women in childbearing age, but it would largely exceed the tolerable values for Hg.

Preovulatory follicle development in goats following oestrous synchronization with progestagens or prostaglandins.

The study reports on differences in the dynamics of growth and functionality of preovulatory follicles in response to oestrous synchronization, either by the administration of two doses of prostaglandin or by an intravaginal progestagen sponge, in goats. The progestagen-treated group (n = 8) showed more follicles of preovulatory size (> or =5.5 mm) than the cloprostenol group (n = 8) during the follicular phase (4.5 +/- 0.6 vs 1.9 +/- 0.2, p < 0.01). The diameters of the largest follicles (LF1, LF2 and LF3) were also larger in the progestagen group (LF1, 7.8 +/- 0.3 vs 7.0 +/- 0.2 mm, p < 0.05; LF2, 6.7 +/- 0.2 vs 5.6 +/- 0.2 mm, p < 0.01; LF3, 5.5 +/- 0.3 vs 4.2 +/- 0.2 mm, p < 0.01). The study of the preovulatory follicles showed that 27.2% (3/11) of the follicles were in the static phase in the cloprostenol group, whilst 71.4% (10/14) were static in progestagen group (p < 0.05). Higher plasma oestradiol levels were recorded in the progestagen-treated goats during the 48 h prior to cloprostenol injection or progestagen withdrawal (4.2 +/- 0.4 vs 3.0 +/- 0.2 pg/ml, p < 0.05). In conclusion, goats with oestrus synchronized by progestagen showed a higher number of preovulatory-sized follicles, but a decreased oestradiol secretion when compared with does with oestrus synchronized by using prostaglandin analogues. These would support the development of alternative protocols for assisted reproduction.

Timing of preovulatory LH surge and ovulation in superovulated sheep are affected by follicular status at start of the FSH treatment.

The aims of this study were to evaluate the chronology of periovulatory events (oestrus behaviour, LH surge and ovulation) in 16 superovulated Manchega sheep and to determine whether follicular status at start of the FSH supply might affect their occurrence. Mean timing for onset of oestrus behaviour was detected at 28.1 +/- 0.7 h after sponge withdrawal; the preovulatory LH surge and ovulation started at 37.2 +/- 0.7 h and 65.4 +/- 0.7 h after progestagen withdrawal, respectively. The intervals between oestrus, LH surge and ovulation were affected by a high individual variability, which might be the cause for reported decreased efficiency in embryo production. Current results also addressed the role of follicular status at start of the superovulatory treatment on the preovulatory LH surge and the ovulation. The interval LH surge-ovulation was increased in ewes with a growing dominant follicle at starting the FSH treatment (32.3 +/- 0.9 vs 28.6 +/- 0.5 h, p < 0.05). The developmental stage of the largest follicle at starting the superovulatory treatment also affected occurrence of LH surge and ovulation; follicles in growing phase advanced the occurrence of the LH surge and ovulation when compared to decreasing follicles (33.0 +/- 1.0 vs 43.5 +/- 1.1 h, p < 0.05, for LH peak and 60.7 +/- 1.1 vs 72.8 +/- 1.2 h, p < 0.05, for ovulation). Thus, only ewes with growing follicles ovulated prior to 55 h after sponge withdrawal; conversely, no sheep with decreasing follicles ovulated earlier than 67 h, when an 85.7% of the ewes bearing growing follicles has ovulated at 63 h.

Influence of vehicle on kinetics of exogenous progesterone administered either by subcutaneous and intramuscular routes to sheep.

This study aimed to determine the best vehicle and administration route for progesterone administration in sheep. In a first replicate, single intramuscular doses of 25mg progesterone were administered to ewes previously ovariectomized, either in propylene glycol (group IM-PG, n=6) or olive oil (group IM-OO, n=5). In a second replicate, the same solutions of progesterone were administered subcutaneously to the same ewes (groups SC-PG, n=6, and SC-OO, n=5). In the present study, the best pharmacokinetic results of a single dose of 25mg of progesterone were obtained, both using PG and OO as vehicles, by the subcutaneous route. Thus, progesterone remained in plasma for a longer time after subcutaneous administration in PG than in OO (t(1/2beta): 60.65+/-13.07 vs. 27.51+/-3.59 h; P<0.05); the mean residence time being higher in SC-PG than in SC-OO group (88.99+/-18.36 vs. 41.04+/-5.31h; P<0.05). However, both vehicles allowed maintained plasma levels 0.5 ng/ml for at least 30 h, so any of these treatments may be efficiently used for administration of exogenous progesterone.

Effects of breed on follicular dynamics and oestradiol secretion during the follicular phase in sheep.

Current study evaluates effects from breed and management background on follicular dynamics and endocrine output during the follicular phase of sheep oestrous cycle. Follicular phases were synchronized with three cloprostenol doses, 10 days apart, in three groups of 10 females of different non-prolific Spanish breeds (Manchega, Rubia del Molar and Negra de Colmenar). Development of all follicles reaching antral diameters >or=2 mm was assessed by daily transrectal ultrasonographies, whereas follicular function was evaluated by measurement of plasma oestradiol concentrations. All the ovulatory follicles were present at induced luteolysis in Manchega sheep, while a 93.7% were detected in Rubia del Molar and Negra de Colmenar ewes. The mean size of these ovulatory follicles was similar between breeds at 0 h, but their growth rates were higher in Manchega ewes, reaching a larger size at oestrous detection than in Negra de Colmenar and Rubia del Molar sheep (p < 0.05). Conversely, the oestradiol levels increased with time in Rubia del Molar and Negra de Colmenar (p < 0.05); whilst remained stable in Manchega females. However, the patterns of follicular turnover were similar between breeds. These results indicate that, though differences in follicular size and size distribution, patterns of follicular turnover in sheep are affected neither by the breed nor by the background of management and selection.

Effects of growth hormone and gonadotrophin releasing hormone antagonists on ovarian follicle growth in sheep.

Our objective was to determine the effects of the administration of growth hormone (GH) alone or plus teverelix, a gonadotrophin releasing hormone antagonist (GnRHa), on follicle development in sheep. Ewes were treated daily for 6 days by the intramuscular route with 15 mg of GH alone (GH group; n = 6) or combined with two subcutaneous doses of GnRHa (1.5 mg) on days 0 and 3 of GH treatment (GH/GnRHa group; n = 6); the control group (n = 6) received similar treatment with saline solution. Plasma follicle stimulating hormone levels were significantly lower in the GH/GnRHa group than in the control (P < 0.001) and GH groups (P < 0.05). The number of follicles > or =2 mm increased to reach significant differences with control (18.7 +/- 0.6) on day 4 in GH/GnRHa group (22.7 +/- 0.5, P < 0.001) and on day 5 in GH group (20.3 +/- 0.4 vs. 17.0 +/- 0.6, P < 0.05). These results indicate that GH and GnRHa may be useful for increasing the number of gonadotrophin-responsive follicles in the ovary. However, follicle function could be affected as both GH and GH/GnRHa groups showed lower plasma inhibin A concentrations than control sheep (90-110 pg/mL vs. 170-185 pg/mL, P < 0.005).

Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep.

Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32 mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4h post-administration, maximum plasma FSH concentration, C(max), and both the area under the plasma concentration-time curve extrapolated to the infinite, AUC(inf), and to the last moment of sampling, AUC(last)) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3 x 1.32 mg, 2 x 1.10 mg, and 3 x 0.88 mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7+/-0.6 versus 10.0+/-0.4 in N and 9.8+/-0.6 in M, P<0.05 for both) and embryos (7.9+/-0.8 versus 4.3+/-0.4 in N, P<0.05, and 6.7+/-0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54+/-0.08 ng/ml for R, 0.45+/-0.05 ng/ml for N and 0.35+/-0.05 ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4+/-2.2), M (18.9+/-1.5, n.s.) and N sheep (14.1+/-1.4; P<0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment.

Administration of single short-acting doses of GnRH antagonist modifies pituitary and follicular function in sheep.

Current study determined the effect of two different single subcutaneous doses (1.5 and 3 mg) of GnRH antagonist (GnRHa) on pituitary and follicular function in non-lactating cyclic ewes. Both doses abolished the pulsatile secretion of luteinizing hormone (LH) for at least 3 days and decreased mean LH concentration during 6 days (0.64 +/- 0.09 for control and 0.54 +/- 0.05, P < 0.005, and 0.46 +/- 0.02, P < 0.00001, for 1.5 and 3 mg, respectively). Supply of GnRHa decreased the number of large dominant follicles, so the total number of smaller follicles, 2-3 mm in size, increased in both treated groups from day 0, reaching its maximum at day 2 in ewes treated with 1.5 mg (19.83+/-1.05 versus 5.83 +/- 0.50 in the control, P < 0.005) and at day 4 in sheep treated with 3mg (18.67 +/- 0.65 versus 5.50 +/- 0.65 in the control, P < 0.0001). However, the analysis of follicular function in terms of inhibin A indicated a possible effect of the higher dose of GnRHa on follicular function. The pattern of inhibin secretion in the group treated with 3mg of GnRHa decreased after the first 48 h, reaching its lowest value on day 4.5 (182.59 +/- 3.75 to 140.28 +/- 9.91 pg/ml, P < 0.05) concentration significant lower than control sheep (171.93 +/- 6.21 pg/ml, P +/- 0.01) or treated with 1.5 mg (168.04 +/- 7.16 pg/ml, P+ /- 0.05). Hence, the use of 1.5 mg would be more suitable to induce the presence of a high number of follicles able to grow to preovulatory sizes.