Utilizzo dell'ultrasonografia nei piccoli ruminanti per la caratterizzazione della dinamica follicolare al fine di identificare fattori ovarici in grado di influenzare la qualità dell'oocita

The current Doctoral Thesis studied the effect of the ovarian status, as evaluated by ultrasonography, on oocyte developmental competence in small ruminants. The first experiment assessed differences in oestrus cycle characteristics, follicle growth in response to exogenous FSH administration, hormonal milieu and oocyte quality in aged and adult sheep. Results obtained evidenced that aging in the sheep is accompanied by alterations in follicle functions, but fertility seems to be maintained, as evaluated by oocyte in vitro developmental competence after FSH stimulation. The second experiment examined the effects of melatonin on ovarian follicular growth patterns and on in vitro oocyte developmental competence in anestrous goats. Melatonin treatment prior to oocyte collection hastened dominant follicle growth phase, thus increasing ovarian follicle turnover, and improved oocyte quality. The third experiment determined influence of the glucogenic supply on follicular growth patterns and oocyte developmental competence in Sarda sheep. Short-term energy inputs modified the growth dynamics of ovarian follicles by increasing both their recruitment and development to larger categories, and improved oocyte developmental competence. In conclusion, this study evidenced the strong link existing between follicle function and oocyte developmental competence and set the basis for the development of new protocols aimed at improving oocyte quality in small ruminants

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation

Exogenous melatonin positively influences follicular dynamics, oocyte developmental competence and blastocyst output in a goat model

The role of melatonin in modulating mammalian reproduction is of particular interest; however, its effects on ovarian follicles and their oocytes still remain to be characterized. This study determined the influence of melatonin treatment on follicular growth patterns and on in vitro oocyte developmental competence. In a first experiment, the effects of melatonin supplementation on follicular dynamics were evaluated using daily transrectal ultrasonographies for 21 days, in 7 multiparous Sarda goats receiving a subcutaneous implant of 18 mg of melatonin and in 5 control untreated does. Melatonin caused more follicular waves (5.2 ± 0.2 versus 4 ± 0.3; P &lt; 0.05) as the waves were shortened at around 2 days when compared with the non-melatonin treated control goats (P &lt; 0.001). Oocyte developmental competence was evaluated in a second experiment by applying procedures for in vitro embryo production. There were no significant differences in the total number of oocytes obtained from 6 control (n = 192) and 7 melatonin-treated (n = 265) goats given follicle stimulating hormone to induce follicular development. Differences in oocyte developmental competence between the two groups became evident after in vitro fertilization and culture; melatonin increased the rate of cleaved oocytes in comparison with control animals (82.5 versus 63.4%; P &lt; 0.001), advanced timing of embryo development and enhanced blastocyst output (31.5 versus 16.3%; P &lt; 0.01). However, blastocyst quality, as evaluated by cryotolerance and gene expression analysis, was not found to be different between the groups. In conclusion, in vivo melatonin treatment is beneficial for increasing ovarian follicle turnover and improving oocyte developmental competence and kinetics of the blastocyst

Effect of aging on follicular function may be relieved by exogenous gonadotropin treatment in a sheep model

The current study investigated hormonal and ovarian changes during physiological reproductive aging in Sarda ewes. In a first experiment, follicular and corpus luteum dynamics were compared during an induced oestrus cycle in aged (12-14 years) and young adult ewes (4-5 years). Oestrus cycle characteristics did not differ between the two experimental groups. However, follicular function during the follicular phase showed significant alterations in aged ewes, as determined by a lack of dominance effect and by lower mean values of circulating oestradiol (E2) and inhibin levels, compared with young adult ewes. In a second experiment, differences in follicle growth, hormonal milieu and oocyte quality in response to exogenous FSH administration were assessed in aged and adult ewes. No differences were recorded in ovarian response to FSH treatment between young adult and aged ewes, as evaluated by ultrasonographic data and circulating concentrations of LH, E2 and inhibin-A. Although the total number of recovered oocytes was similar in the two age groups, the number of good quality oocytes selected for IVM was significantly lower in aged ewes compared with adult ones. Thereafter, no differences were recorded in cleavage rates, total blastocyst output, embryo developmental kinetic and quality between aged and adult groups. In conclusion, this study demonstrated that reproductive aging in sheep is associated with impaired follicle functionality and an increase in the proportion of oocytes showing morphological abnormalities. However interestingly, oocyte developmental competence in vitro and embryo cryotolerance were not affected by the aging process, when only good quality oocytes were chosen. © 2012 Society for Reproduction and Fertility

Changes in renal hemodynamics of undernourished fetuses appear earlier than IUGR evidences

The present study used a sheep model of intrauterine growth restriction, combining maternal undernutrition and twinning, to determine possible markers of early damage to the fetal kidney. The occurrence of early deviations in fetal hemodynamics which may be indicative of changes in blood perfusion was assessed by Doppler ultrasonography. A total of 24 sheep divided in two groups were fed with the same standard grain-based diet but fulfilling either their daily maintenance requirements for pregnancy (control group; n=12, six singleton and six twin pregnancies) or only the 50% of such quantity (food-restricted group; n=12; four singleton and eight twin pregnancies). All the fetuses were assessed by both B-mode and Doppler ultrasonography at Day 115 of pregnancy. Fetal blood supply was affected by maternal undernutrition, although there were still no evidences of brain-sparing excepting in fetuses at greatest challenge (twins in underfed pregnancies). However, there were early changes in the blood supply to the kidneys of underfed fetuses and underfed twins evidenced decreases in kidney size

Glucogenic supply increases oocyte developmental competence in sheep

The present study aimed to determine the influence of a glucogenic supply on oocyte developmental competence. Oestrous cycles were synchronised in 22 Sardaewes by the insertion (Day 0) of one intravaginal progestagen-impregnated sponge that was removed after 6 days. After removal, the ewes were randomly allocated into two experimental groups (treated and control ewes) and, from Day 7 to Day 11, treated ewes received oral administration of a glucogenic mixture, whereas control animals received water. Follicular development was stimulated by FSH administration from Days 8 to 10. Glucose metabolism was assessed from Days 7 to 11, whilst follicle and corpus luteum growth dynamics and functionality were evaluated between Days 6 and 11. At Day 11 ovaries were collected and processed for in vitro embryo production. Glucogenic treatment increased both the plasma levels of glucose, progesterone, oestradiol and the number of 2-3-mm follicles (P < 0.05). Higher fertilisation and blastocyst rates (P < 0.05) were obtained after IVM of oocytes recovered from treated ewes compared with control ones. In conclusion, glucogenic treatment modifies follicle and corpus luteum functionality and improves oocyte quality, as evaluated by in vitro developmental kinetics and blastocyst output. © CSIRO 2012

<i>In vitro</i> production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment

This study compares the developmental capacity and cryotolerance of embryos produced from oocytes of stimulated prepubertal and adult Sarda goats. Twelve prepubertal and 13 adult goats were each given 110 and 175 IU FSH, respectively, and cumulus–oocyte complexes (COCs) were collected by laparoscopic oocyte-pick-up (LOPU). After in vitro maturation, fertilisation and culture (IVMFC), blastocysts were vitrified, warmed and blastocoel re-expansion and gene expression were evaluated. Prepubertal goats produced a higher COCs number than adults (mean ± s.e.m., 89.67 ± 5.74 and 26.69 ± 3.66, respectively; P &lt; 0.01). Lower developmental competence was demonstrated in the prepubertal oocytes as shown by a higher number of COCs discarded before IVM (21.1% and 14.7% for prepubertals and adults, respectively; P &lt; 0.01) and IVF (23.4% v. 9.1%; P &lt; 0.01) and by the lower cleavage (55.6% and 70.3%, respectively; P &lt; 0.01) and blastocyst rates (24.2% and 33.9%, respectively; P &lt; 0.05). Compared with the adult, prepubertal vitrified/warmed blastocysts showed significantly (P &lt; 0.05) lower in vitro viability, as determined by the re-expansion rate (62.5% and 40.3%). No differences were observed in the time required for blastocoel re-expansion or in cyclin B1, E-cadherin, Na/K ATPase, HSP90β and aquaporin 3 messenger RNA quantity. These results show that in vitro-produced embryos produced from prepubertal goat oocytes have a lower developmental rate and cryotolerance compared with their adult counterparts. However, we can assume that the quality of re-expanded embryos does not differ between the two groups

Melatonin deprival modifies folliclar and corpus luteal growth dynamics in a sheep model

This study assessed the effect of melatonin deprival on ovarian status and function in sheep. Experimental procedures were carried out within two consecutive breeding seasons. Animals were divided into two groups: pinealectomized (n=6); sham-operated (n=6). The completeness of the pineal gland removal was confirmed by the plasma concentration of melatonin. Ovarian status was monitored by ovarian ultrasonography during one year to study reproductive seasonality. Follicles and corpora lutea growth dynamics were assessed during an induced oestrous cycle. Since melatonin effects on the ovary may be also mediated by its antioxidant properties, plasma trolox equivalent antioxidant capacity (TEAC) was determined monthly during one year. Pinealectomy significantly extended the breeding season (310 ± 24.7 vs 217.5 ± 24.7 days in controls; p<0.05). Both pinealectomized and sham-operated ewes showed a well-defined wave-like pattern of follicle dynamics; however, melatonin deficiency caused fewer waves during the oestrous cycle (4.3 ± 0.2 vs. 5.2 ± 0.2; p<0.05) because waves were one day longer when compared with the controls (7.2 ± 0.3 vs. 6.1 ± 0.3; p<0.05). The mean area of the corpora lutea (105.4 ± 5.9 vs. 65.4 ± 5.9 mm2; p<0.05) and progesterone plasma levels (7.1 ± 0.7 vs 4.9 ± 0.6 ng/mL; p<0.05) were significantly higher in sham-operated compared with pinealectomized ewes. In addition, TEAC values were significantly lower in pinealectomized ewes compared to control ones. These data suggest that melatonin, besides exerting its well-known role in the synchronization of seasonal reproductive fluctuations, also influences the growth pattern of the follicles and the steroidogenic capacity of the corpus luteum

Postnatal pituitary and follicular activation: A revisited hypothesis in a sheep model

The importance of postnatal pituitary activation as regards female reproductive development is not yet understood.By taking advantage of the experimental model developed in a previous study, i.e. ewe lambs expressing markedly different ovarian phenotypes at 50 days of age, we design this study to determine whether differences found in ovarian status during the early prepubertal period are due to different patterns of postnatal pituitary activation, and to assess whether these differences have long lasting effects on subsequent reproductive performance. Results showed that ewe lambs with high antral follicle count (AFC) at 50 days of age had significantly lower plasma follicle stimulating hormone (FSH) concentrations and higher anti-Mullerian hormone (AMH) concentrations during the first 9 weeks of age compared with low AFC ewe lambs (p<0.0001). With a longitudinal experiment we showed that a high AFC in the early prepubertal period is associated with consistently higher AMH concentrations and number of antral follicles up to the post-pubertal period, and with higher pregnancy rates in the first breeding season. In addition, the effect of age in decreasing AMH concentrations was more marked in the low AFC group. Results of the present study demonstrate that ewe lambs undergo different patterns of postnatal pituitary activation. A high AFC at 50 days of age indicates an advanced phase of ovarian maturation, which was accompanied by constantly higher AMH concentrations up to the post-pubertal period, a greater ovarian response to FSH stimulation and by higher pregnancy rates at first mating, as compared with the low AFC group