Sperm quality in wild-caught and farmed males of the European eel (Anguilla anguilla).

Sperm density and performance of wild and farmed male European eels were evaluated to investigate the effect of maintenance in captivity on ejaculate quality. Hormonal stimulation (with human chorionic gonadotropin) lasted for 17 weeks. Different origins of the male European eels partially influenced the quality of the ejaculates. Indeed, wild animals (i.e., collected at a natural site) had greater sperm longevity during the hormonal stimulation, whereas there was no significant effect of the origin on sperm density, spermatocrit, the percentage of motile sperm, and plasma testosterone concentrations. The different origins of the males also affected fatty acid sperm content, with greater unsaturated fatty acid and omega-6 concentrations in wild eels and lesser concentrations of saturated fatty acids and omega-3 fatty acids in farmed eels. Regarding sperm quality of European eels, this is the first study that takes into account the effect of different origins of stimulated males (wild-caught compared with farmed) on sperm quality, and these findings may help to improve the production of high-quality gametes in this endangered species.

Effect of photoperiod on endocrine profiles and vitellogenin expression in European eels Anguilla anguilla during artificially induced ovarian development.

The aim of this work was to determine the effects of dark and light conditions on the E2, testosterone and thyroid hormones levels and on the gene expression levels (vitellogenin 1, vitellogenin 2, and estradiol receptor one) in European eels (Anguilla anguilla) during ovarian development induced by increasing doses of carp pituitary extracts (CPEs). The subjects were divided into 2 groups: 14-hour light:10-hour dark (Light Group) and 24-hour darkness (Dark Group). All the eels received intramuscular injections with CPE at a dosage of 10 mg/kg body weight (BW) once a week for the first 3 weeks, 20 mg/kg BW fourth-sixth week, 30 mg/kg BW seventh-ninth week, and 40 mg/kg up to the end of the experiment (13th week). Vitellogenin and estradiol receptor expression levels did not show significant differences between the two housing conditions whereas in both groups vitellogenin mRNA increased starting from first CPE injection. Testosterone and 17-beta estradiol plasma levels were significantly greater in the Dark Group compared with the Light Group starting from the ninth and the 13th week, respectively. These results suggest that darkness could be a useful variable for standardizing gonadal maturation in eels kept in captivity.

Effects of repeated transvaginal ultrasound-guided aspirations performed in anestrous and cyclic mares on P4 and E2 plasma levels and luteal function.

The aim of the present study was to verify how repeated ovum pick-up (OPU), performed in anestrous and cyclic mares, affect ovarian activity, measured by progesterone (P4) and 17ß-estradiol (E2) plasma levels. Ovum pick-up of all visible follicles was performed every 9 to 12 days, and four sessions were carried out during anestrous (A) and breeding season (BS). The number of aspirated follicles per mare at each session was not significantly different between the two periods (BS: 6.1 ± 2.4; A: 7.5 ± 4.4; P > 0.05), but the mean follicular diameter was significantly higher during BS (16.0 ± 7.1 vs. 10.2 ± 5.1 mm; P < 0.05); during A the number of aspirated follicles less than 15 mm in diameter resulted significantly higher than that registered in BS (5.1 ± 2.7 vs. 3.0 ± 1.8; P < 0.05). The total mean value of P4 was higher in BS than in A (6.3 ± 4.4 vs. 0.3 ± 1.8 ng/mL; P < 0.05), whereas the total mean level of E2 was not different between the two periods (3.8 ± 3.4 vs. 2.5 ± 2.7 pg/mL; P > 0.05). Estradiol plasma values resulted positively correlated, in A and BS, with diameter of follicles detected on the ovaries (R = 0.345 and R = 0.331, respectively), whereas a negative correlation was observed between P4 and follicular diameter in BS (R = -0.162). Both E2 and P4 presented a high individual variability during BS; in particular, in three of seven mares, P4 trend was compatible with a normal estrous cycle, and the interval between two consecutive peaks was 21 days. In two of seven mares, with CL at first OPU, P4 concentrations remained more than 3 ng/mL throughout the entire treatment period. Finally, in two of seven animals, P4 levels initially showed a similar pattern to that of a normal estrous cycle, then, after the second aspiration, they remained consistently higher than 3 ng/mL. When the procedure was carried out in cyclic animals, the influence of this technique on ovarian activity seemed to be related to individual variability although, according to progesterone values, structures observed on the ovaries after aspirations presented luteal function. Furthermore, the resumption of normal ovarian activity, after repeated OPU sessions, occurred in a period not much longer than the duration of a normal estrous cycle (25.4 ± 5.2 days). Data recorded during nonbreeding period showed that repeated OPU in anestrous mares do not affect ovarian activity and do not anticipate the resumption of ovarian cyclicity. However, based on the number of aspirated follicles in anestrous and cyclic mares, both types of subjects could be considered as oocyte donors.

A study on some welfare-related parameters of hDAF transgenic pigs when compared with their conventional close relatives.

Pigs are increasingly used in medical research as transgenic laboratory animals; however, little knowledge is presently available concerning their welfare assessment. The aim of the present study was to investigate some welfare-related parameters of transgenic pigs intended for xenotrasplantation (human decay-accelerating factor (hDAF)) when compared with their conventional (i.e. not transgenic) close relatives (full sibs and half sibs). A total of 14 Large White female transgenic pigs and 10 female non-transgenic (conventional) pigs from four litters were used. All pigs were from the same conventional boar, donor of the semen treated for sperm-mediated gene transfer. During the experiment, BW ranged from 50 to about 80 kg and pigs were weighed at the beginning and at the end of the experiment. Animals were subjected to a set of behavioural tests: a human approach test (HAT), a novel object test (NOT) and an open-door test (ODT). Food preferences were tested through the offer of different foods (banana, apple, carrot, cracker and lemon). During a 4-day period, pigs were diurnally videotaped to study the prevalence of the different behaviours and social interactions (aggressive and non-aggressive interactions). At the end of the trial, cortisol level had been assessed on bristles. No significant differences (P>0.05) were observed between hDAF transgenic and conventional pigs with respect to growth traits, reactivity towards unexpected situations (HAT, NOT, ODT), food preferences, main behavioural traits, social interactions and hair cortisol.

Carry-over effects of body condition in the early pregnant ewe on peri-partum adipose tissue metabolism.

This study evaluated the carry-over effects of ewe body reserves during early pregnancy on peri-partum adipose tissue metabolism. Forty-nine multiparous ewes were divided in three categories according to their body condition score (BCS) at day 30 of pregnancy (BCS < 3, 2.5-2.75; BCS = 3; BCS > 3, 3.25-3.5). Live-weight (LW) and BCS gains from 1st to 4th month of pregnancy were greater in ewes with BCS < 3 and 3 than in >3 animals. In contrast, in the last month of pregnancy, there was BCS decrease in all groups, although LW continued increasing. There were no differences in LW or BCS across ewe categories during this period. Peripheral leptin levels throughout the three last weeks of pregnancy were greater in ewes with BCS > 3 than in the rest, but this difference did not persist after lambing. Plasma metabolites related to energy metabolism, milk yield and lamb growth were not affected by ewe BCS in early pregnancy. Long-chain saturated milk fatty acids (FA) (C16-C24) were greater in ewes with lowest BCS (<3 and 3). Ewes with greater BCS showed greater monounsaturated and lowest polyunsaturated milk FA content. Ewe post-mating body reserves affect both pre-partum leptinaemia and post-partum milk polyunsaturated fatty acids content, but it had little effect on lamb performance.

Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome.

Embryo transfer (ET) in the donkey resulted in a very low recipient pregnancy rates. The aim of these studies was to investigate if nonsurgical transfer techniques or donkey embryo quality affect donkey recipient pregnancy failure. In Study 1, the impact of transfer technique was investigated by evaluating if cervical catheterization is associated with prostaglandin release and suppression of luteal function and if donkey recipients would become pregnant after nonsurgical transfer of horse embryos. Four jennies, from 5 to 8 d after ovulation, were submitted to a sham transcervical ET and to evaluation of PGFM and progesterone plasma concentrations. Five 8 d horse embryos were nonsurgically transferred into synchronized donkey recipients (HD). Cervical stimulation caused a transient PGF(2α) release in two of four jennies in the absence of a significant decrease in progesterone plasma concentration. All transferred horse embryos resulted in pregnancies in the jenny recipients. In Study 2, donkey embryo viability was investigated by 1.2 meters, 6-diamidino-2-phenylindole (DAPI) staining of 10 embryos and by the transfer of 6 and 12 donkey embryos in synchronized mare (DH) and donkey (DD) recipients, respectively, of known fertility. The estimated proportion of dead cells in DAPI stained embryos was 0.9% (range 0-3.9%) and below what is considered normal (20%) for horse embryos. Three of six and six of 12 of the DH and DD ETs, respectively resulted in pregnancies at 14 and 25 d (50%), a higher pregnancy rate than previously reported after DD ET. The overall results of this study suggest that the transcervical technique for ET and donkey embryo viability are not the reasons for the low pregnancy rates that have previously been described in donkey recipients, and that nonsurgical ET in donkeys can result in acceptable results.

Oxytocin, vasopressin, prostaglandin F(2alpha), luteinizing hormone, testosterone, estrone sulfate, and cortisol plasma concentrations after sexual stimulation in stallions.

This experiment was designed to determine the effects of sexual stimulation on plasma concentrations of oxytocin (OT), vasopressin (VP), 15-ketodihydro-PGF(2alpha) (PG-metabolite), luteinizing hormone (LH), testosterone (T), estrone sulfate (ES), and cortisol (C) in stallions. Semen samples were collected from 14 light horse stallions (Equus caballus) of proven fertility using a Missouri model artificial vagina. Blood samples were collected at 15, 12, 9, 6, and 3 min before estrous mare exposure, at erection, at ejaculation, and at 3, 6, and 9 min after ejaculation. Afterwards, blood sampling was performed every 10 min for the following 60 min. Sexual activity determined an increase in plasma concentrations of OT, VP, C, PG-metabolite, and ES and caused no changes in LH and T concentrations. The finding of a negative correlation between C and VP at erection, and between C and T before erection and at the time of erection, could be explained by a possible inhibitory role exerted by C in the mechanism of sexual arousal described for men.

Administration of sulpiride or domperidone for advancing the first ovulation in deep anestrous mares.

Since results with using sulpiride and domperidone are conflicting and since both have not been tested at the same time, the aim of this study was to compare the efficacy of these substances for the induction of ovulation in deep anestrous mares in the same experimental conditions and to determine their fertility after artificial insemination (AI) at the induced estrus. Twenty-six non-pregnant, non-lactating standardbred anestrous mares were randomly assigned to three groups and treated daily for 25 days (from February 3rd to February 28th) with either sulpiride (1mg/kg of body weight im SID, n=10), or domperidone (1mg/kg po SID, n=10); 6 animals were used as control. The beginning of the transition period and the first ovulation were hastened in sulpiride (16.4+/-0.8 days) but not in domperidone (46.0+/-3.3 days) treated mares (P<0.05). The diameter of the largest follicle was affected by treatment, time and interaction of treatment-by-day (P<0.05) and significantly increased in the sulpiride group (P<0.05). Although a main effect of treatment on plasma LH concentration was not observed (P=0.06), time and interaction of treatment-by-day were statistically significant (P<0.05). The interval from the beginning of treatment to first ovulation was shorter (P<0.05) in the sulpiride group (36.9+/-2.5 days) than in the domperidone (74.7+/-3.3 days) and control (81.4+/-3.1) groups. The establishment of pregnancy was significantly (P<0.05) hastened in sulpiride (61.0+/-35.2 days) but not in domperidone (83.0+/-44.0 days) treated mares. Treated mares not pregnant after the first AI, showed normal estrous cycles with regular interovulatory intervals (P>0.05). It was concluded that sulpiride is effective in advancing the beginning of transition period and the first ovulation whereas domperidone is successful only in some mares.

15-Ketodihydro-PGF2alpha and cortisol plasma concentrations in newborn foals after spontaneous or oxytocin-induced parturition.

Hormonal changes during early neonatal life play a major role in the physiological processes underlying the maturation of several organs. Since prostaglandins and cortisol are associated with fetal organ system maturation, the aim of this study was to evaluate 15-ketodihydro-PGF(2alpha) (PGM) and cortisol plasma concentrations during the first 21 days after birth in foals born by either spontaneous (24 foals) or low-dose oxytocin (OT)-induced parturition performed after at least 320 gestational days (25 foals) since induction is often considered to be a cause of prematurity. After spontaneous birth, the PGM concentration was significantly (P<0.05) higher at 20 and 30min compared to samples taken several hours or days later, while induced foals showed significantly (P<0.05) higher concentrations at 10, 20, and 30min. Regarding differences between the two groups, the plasma concentration of PGM was significantly higher 10 (P<0.01), 20 (P<0.05), and 30 (P<0.05)min and 3h (P<0.05) after birth in induced foals compared to foals born by spontaneous parturition. It is difficult to determine whether the higher initial PGM concentrations in induced foals is related to higher uterine or fetal PGM release induced by exogenous OT stimulation. Cortisol plasma levels in both groups were higher at birth (P<0.05) compared to the later sampling times. No differences were observed between the two groups indicating that the induction protocol used does not seem to result in premature foals.

Embryo production in superovulated goats treated with insulin before or after mating or by continuous propylene glycol supplementation.

Seventeen adult and cyclic Moxoto goats were synchronized using 60 mg MPA vaginal sponge for 11 days and 50 mug cloprostenol, 48 h before sponge removal, and superovulated with 120 mg pFSH i.m. in decreasing doses at 12 h intervals for three consecutive days. In seven goats, 0.2 IU/kg BW/day of long acting insulin was subcutaneously injected at same time as pFSH, and in the other five goats, the same dose of insulin was injected for three consecutive days starting 24 h after mating. Finally, five goats were supplemented with an oral dose of 80 ml/goat/day of propylene glycol continuously during the experiment. The animals were flushed at 7 days after mating and the embryos were classified based on International Embryo Transfer Society criteria. Blood samples were collected every 3 days for insulin assay. Administration of insulin raised the insulin levels of the goats (p < 0.05), whereas in the group treated with propylene glycol, insulin rate was different only between FSH treatment and after mating (p < 0.05). Similar rates of recovery for total (80.05 +/- 9.78%) or transferable structures (61.03 +/- 15.13%) were obtained. Treatment was not influenced (p > 0.05) by responsiveness to superovulation, which averaged 64%. By contrast, insulin treatments were shown to increase the number of embryos considered excellent with respect to goats supplemented with propylene glycol (p < 0.05). When insulin was given before mating, a strong relationship (r = 0. 90) (p < 0.05) between number of transferable embryo and ovulations was observed in the animals. In conclusion, superovulated goats treated with low doses of exogenous insulin resulted in an enhancement in embryo quality, which was related to changes in circulating insulin concentrations.

Acyl ghrelin and metabolic hormones in pregnant and lactating sows.

Ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor, is considered a pleiotropic regulator involved in a large array of functions, including control of energy balance, regulation of food intake and, more recently, modulation of the reproductive axis. The present study was aimed at determining the changes in plasma concentrations of acyl-ghrelin in pregnant and lactating sows, with special emphasis on the relationship with the levels of GH, leptin, non-esterified fatty acids (NEFA) and insulin-like growth factor (IGF-1). Blood samples were collected via jugular venipuncture from 22 multiparous sow 30, 60 and 90 days after artificial insemination, 7 and 21 days after farrowing and at first oestrus post-weaning. Plasma concentrations of acyl-ghrelin, leptin, GH and IGF-1 were quantified by validated radioimmunoassay; NEFA were determined using a colorimetric procedure. Plasma acyl ghrelin levels were highest at 30 days of pregnancy and decreased thereafter and during lactation. At the beginning of lactation, GH, IGF-1 and NEFA concentrations significantly increased, while a significant reduction occurred in leptin. In conclusion, ghrelin concentrations in sow maternal circulation does not seem to play an important role in maintaining circulating GH levels during lactation; moreover, ghrelin is not associated with leptin, NEFA and IGF-1 levels.

Gastric immunolocalization and plasma profiles of acyl-ghrelin in fasted and fasted-refed prepuberal gilts.

Ghrelin is a peripheral circulating hormone, mainly released from the stomach, which can stimulate food intake. We studied fed, fasted and fasted-refed prepuberal gilts in order to outline possible changes in gastric mucosal ghrelin cells and in plasma ghrelin profiles in response to food deprivation. Acyl-ghrelin-immunoreactive cells were numerous in oxyntic glands, less abundant in cardiac glands and least frequent in pyloric glands, with the addition of a minor population of labelled cells in the gastric pit mucosa. When fed and fasted animals were compared (72-h fast versus fed; n = 4 each), no clear-cut differences were revealed in labelled cell numbers, nor in their staining intensity. An RIA for plasma porcine acyl-ghrelin (n-octanoylated at Ser-3), not recognizing des-acyl-ghrelin, was validated. Plasma acyl-ghrelin progressively increased upon fasting (over 6, 12, 24 and 48 h); ghrelin levels significantly (P<0.05) higher than those prefast were reached at 72 h. After refeeding, plasma ghrelin was rapidly restored to basal values by 6 h. In the same animals, plasma insulin was significantly reduced throughout the fasting period (6-72 h), while rapidly increasing after refeeding. Non-esterified fatty acid levels increased during fasting (12-72 h) and rapidly returned to low values after refeeding. In conclusion, the present study demonstrates that starvation and refeeding influence ghrelin plasma level in prepuberal gilts. The absence of detectable changes in ghrelin cells, as seen in immunohistochemistry, could be due to a large intracellular storage of potentially releasable acylghrelin.

Leptin, GH, PRL, insulin and metabolic parameters throughout the dry period and lactation in dairy cows.

Leptin may play a role in the endocrine-metabolic processes that guarantee the physiological course of lactation in dairy cattle. This study was aimed at determining the changes in plasma concentrations of leptin and some of the main hormones and metabolites involved in the lactogenetic process in high-yielding dairy cows throughout lactation; we also wanted to assess whether leptin secretion is subjected to seasonal influences. Blood samples were collected from 23 Italian Friesian dairy cows from the end of a lactation to the ninth month of the subsequent one; in addition, blood was sampled from 47 dairy cows in different phases of lactation during February and July. Plasma concentrations of leptin, growth hormone (GH), insulin, prolactin (PRL), glucose, non-esterified fatty acids (NEFA) and urea were quantified by either validated radioimmunoassay (RIA) or enzymatic colorimetric methods. At the beginning of lactation, GH concentrations significantly increased, while a significant reduction occurred in leptin and insulin. This endocrine condition, such as the significant increase in NEFA plasma concentrations, is indicative of a marked lipid mobilization. In the more advanced stages of lactation, when both energy and protein balances become positive, leptin plasma concentrations increased, whereas GH and NEFA concentrations declined. During the summer months, a significant increase in leptin plasma concentrations, irrespective of the phase of lactation, was observed. Collectively, our findings suggest that, in dairy cows, leptin may represent a 'metabolic signal' of animal's status of fattening and nutritional level; in addition, leptin seems to be influenced by photoperiod and environmental temperature.

Leptin concentrations in plasma and follicular fluid from prepubertal gilts as influenced by fasting, refeeding and insulin.

This study's aim was to examine whether fasting and refeeding would influence leptin levels in both plasma and follicular fluid from prepubertal gilts, and whether insulin affects leptin levels in fasting gilts. In experiment 1, four gilts were fasted for 72 h and then refed. Blood samples were withdrawn during normoalimentation, at the end of fasting, and for 4 h after refeeding. All samples were assayed for leptin; alternate samples were assayed for insulin, glucose and non-esterified fatty acids (NEFA). Fasting caused a decrease in leptin, glucose and insulin levels in plasma, while NEFA concentrations increased. In experiment 2, four gilts were given insulin as a bolus (0.2 IU/kg body weight) after 68 h of fasting. Blood samples were collected every 15 min around insulin administration and were assayed for leptin, insulin and glucose. This experiment shows that insulin administration increases leptin levels during fasting. In experiment 3, gilts were ovariectomized during normal alimentation (n=4), after 48 h of fasting (n=4), and after 48 h of realimentation following 48 h of fasting (n=4). Leptin levels in both plasma and follicular fluid collected after 48 h of fasting were significantly lower than those observed during normoalimentation or refeeding. In conclusion, a transient increase in insulin during fasting is effective in restoring leptin concentrations; in addition, leptin levels in follicular fluid parallel those in plasma.

Leptin does not seem to influence glucose uptake by bovine mammary explants.

Leptin, a protein produced and secreted by adipocytes, is know to regulate food intake and whole-body energy metabolism, but knowledge about its possible effect in bovine mammary gland is scarce. Leptin may be involved in the regulation of glucose transport even though this effect at the tissue level remains controversial. Once uptaken by the mammary gland, glucose is utilised in several ways but the majority, about 60-70%, is drained for lactose synthesis. This study was aimed at investigating the effect of leptin on glucose regulation in bovine mammary gland. We have examined the effects of leptin on the expression of GLUT1 mRNA, pyruvate kinase (PK) as well as glucose-6-phosphate dehydrogenase (G6PDH) activity. Treatment of mammary gland explants with recombinant leptin did not influence glucose assimilation, pathway transport (GLUT1 mRNA) and glucose metabolism (PK and G6PDH) in this tissue. The results from this study seem to exclude an involvement of leptin in glucose uptake and metabolism in bovine mammary gland.

Stimulatory effects of fasting on vascular endothelial growth factor (VEGF) production by growing pig ovarian follicles.

The aim of this study was to investigate the effect of fasting on both vascular endothelial growth factor (VEGF) production and VEGF mRNA expression in growing ovarian follicles (>5 mm in diameter) from gilts at 48 h after equine chorionic gonadotrophin (eCG) treatment. The concentrations of VEGF and albumin were measured in the follicular fluid of single follicles, and VEGF mRNA was determined in the follicle wall. Fasting resulted in a significant increase in VEGF concentrations in follicular fluid (20.64+/-0.72 versus 10.79+/-0.86 ng ml(-1), P<0.001), but it did not affect the total amount of VEGF mRNA in the follicle wall compared with that of fed animals. However, VEGF mRNA in the theca and granulosa compartments increased and decreased, respectively, compared with that of fed animals. The concentrations of albumin measured in follicular fluid as an index of vessel permeability were higher in fasted than in animals fed normally, most likely as a result of the increased VEGF production. Follicular steroidogenesis was impaired in fasted animals. Progesterone was the most abundant steroid in the follicular fluid and oestradiol was present in lower concentrations, thus indicating an alteration in the steroidogenic enzymatic cascade. In conclusion, fasting induces an increase in both VEGF production and vessel permeability. Such a reaction is unable under severe food deprivation to preserve follicle function, but may represent a mechanism that regulates blood vessel extension and distribution in relation to tissue requirements and availability of systemic nutrient.

5'-deiodinase activity and circulating thyronines in lactating cows.

To investigate the correlation between lactation and thyroid hormone metabolism, the authors studied concentrations of total and free thyroxine (T4 and fT4), triiodothyronine (T3 and fT3), and reverse triiodothyronine (rT3) in plasma and milk, as well as liver and mammary gland 5'-deiodinase (5'D) activity in dry, early, middle, and late lactating dairy cows. Cows in early lactation show lower plasma levels of T4 and rT3 than dry, middle, and late lactating animals, whereas T3 shows the lowest plasma levels in the dry period; free T4 and T3 show a similar pattern. In early lactation there is a clear decrease in liver 5'D associated with a notable increase in mammary 5'D. Concentrations of T4 and T3 in milk drop significantly in the first few days after delivery, whereas rT3 increases up to the fourth month. The findings suggest a relationship between the hypothyroid status of lactating cows and the rearrangement of organ-specific 5'-deiodinase activity related to the maintenance of the udder's function.