Determination of Rhythmicity and Gestational Stage-Related Distribution of Blood Plasma Melatonin Concentrations in Donkey Mares.

The aim of this study is to obtain a more complete picture of blood plasma melatonin concentrations in the donkey mares. To this purpose, sampling and statistical processing were carried out in such a way that allowed the researchers to establish the annual and daily rhythms. Based on human observations, according to the hypothesis of the authors, the blood plasma melatonin concentration of pregnant individuals rises during the late gestational period, before parturition. To confirm this, the melatonin concentrations of pregnant and non-pregnant jennies were monitored and compared. In regard to the circannual rhythm, the significantly lowest midnight melatonin value (27.67 pg mL-1) was typical for the summer solstice. Under consideration of circadian changes, a significantly strongest melatonin production (45.16 pg mL-1) was observed on the night of the winter solstice (p < 0.001). Considering gestational age, the blood plasma melatonin concentration (around 38 pg mL-1) does not change as gestation progresses (p = 0.136). The results obtained in this studied population of the domestic ass usefully expand the little knowledge previously gathered about the development of the blood plasma melatonin concentrations of this species.

Effects of different cryopreservation methods on canine isolated preantral follicles.

The aim of the present study was to compare the survival and developmental rate of canine isolated preantral follicles (PAFs) after cryopreservation with different methods (closed vs open vitrification). Follicles were isolated from ovaries randomly divided into three groups: fresh control, OPS (open pulled straw) vitrified and cryotube (CT) vitrified. Post-thaw viability of follicles and oocytes was assessed. Fresh and vitrified/thawed PAFs were cultured in 20 µl drops of FSH-supplemented medium for 10 days. Follicular growth, survival rate, estradiol production and ovulation rate were examined. CT method resulted in lower rate of live cells (58.7%) and oocytes (38.8%) than that of fresh ones (83.6% and 64%, respectively) and OPS (80.3% and 79.3%, respectively). Survival rate was similar to fresh follicles in OPS group (98.5% and 95.4%, respectively), while CT decreased the survival to 81.2%. Fresh follicles showed continuous growth, while CT follicles stopped to increase their size after 2 day. In the OPS vitrified follicles, this halting occurred between Day5 and Day10. Fresh follicles showed the highest estradiol production (range: 26.9 - 266.2 pg/ml). Comparing the two vitrified groups, lower estradiol concentration range was measured in the CT group (7.8-48.7 pg/ml vs. 15.4-89.6 pg/ml). Ovulation rate in each group was lowest in the OPS group (1.7% vs 7% and 8.9% in fesh and CT, respectively). Our data show that OPS vitrification provides superior survival rate, in vitro growth and hormonal production to CT. To our knowledge, these are the first results on comparing different cryopreservation protocols on canine isolated preantral follicles.

Plasma melatonin concentration during the early post-partum period in Thoroughbred mares and their foals.

The authors aimed to determine the plasma melatonin concentration in mares and their new-born foals in the early post-partum period. Blood samples were collected from the jugular vein of 53 mare-foal pairs within twelve hours after parturition. Plasma melatonin levels were measured by ELISA. The melatonin concentration, adjusted for the moment of parturition using a generalised linear model, was 34.58 pg mL-1 in mares. It was significantly lower (27.63 pg mL-1) in the new-born foals. However, the melatonin concentration declined differently by the end of the twelve hours, it decreased less in the offspring than in the mothers. An artificial light supplementation at the end of gestation reduced the melatonin concentration both in mares and their foals by about 10 pg mL-1, compared to the controls. An elevated melatonin production may be related to preparation of mares for parturition and ensures the chances of survival of offspring, therefore the melatonin may reach its peak at the moment of foaling regardless of its actual time. The effect of low melatonin concentration in new-born foals might be associated with the foal's health and subsequent performance. The need to monitor the melatonin concentration in the offspring justifies further studies.

Effect of pituitary adenylate cyclase-activating polypeptide supplementation, applied during or after vitrification on mouse embryo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. It occurs in high levels in the gonads suggesting a potential central role in reproduction. The aim of our study was to assess the effect of PACAP treatment during embryo vitrification on the developmental rate and the expression of the heparin-binding EGF-like growth factor gene (Hbegf). Mouse embryos, obtained from superovulated females were allocated into the four treatment groups. In EM1 and EM2, the embryos were prepared for vitrification in an Equilibration Solution that was supplemented with 1 or 2 µM PACAP1-38, respectively. The embyos in groups CM1 and CM2 were not treated prior to vitrification but were cultured in a medium supplemented with 1 or 2 µM PACAP1-38 after thawing. The Vitrified Control group consisted of embryos vitrified and thawed then cultured without PACAP1-38 treatment. A non-vitrified, non-treated Fresh Control group was also used. After 24 h of culture, the developmental rate of the embryos, as well as the relative expression level of the Hbegf gene, as determined by qPCR, were compared among groups. Higher developmental rate and Hbegf gene expression level were found in the embryos treated with a higher concentration of PACAP. These results indicate that PACAP treatment has a beneficial effect on the survival and development of vitrified/thawed mouse embryos.

Post-thaw viability of mouse preantral follicles after cryopreservation with cryotube freezing and OPS vitrification procedures.

In the field of reproductive science, there is an increased interest in the application of ovarian preantral follicles. Since the ovary contains a great amount of preantral follicles (PAF), the cryopreservation and in vitro culture of such follicles support the fertility preservation of domestic animals with high genetic value, endangered or zoo animals, and women before anticancer therapy. To date, no standard freezing or vitrification protocol is available in human or animals. The aim of the present study was to examine the viability of preantral follicles cryopreserved using freezing or vitrification protocols: cryotube freezing or OPS vitrification.

Anti-Müllerian hormone, testosterone, and insulin-like peptide 3 as biomarkers of Sertoli and Leydig cell function during deslorelin-induced testicular downregulation in the dog.

The role of anti-Müllerian hormone (AMH) and insulin-like peptide 3 (INSL3) in male infertility is not fully understood. We used the downregulated testis as a model of gonadotropin-dependent infertility. Serum testosterone and AMH concentrations were studied in five adult male Beagles implanted (day 0) with 4.7 mg deslorelin (Suprelorin®, Virbac) (DES group). Testicular expression of LH receptor (LHR) and androgen receptor (AR), AMH, type 2 AMH receptor (AMHR2), INSL3 and its receptor (RXFP2) was evaluated 112 days (16 weeks) after deslorelin treatment by qPCR and immunohistochemistry, and compared to untreated adult (CON, n = 6) and prepubertal (PRE, n = 8) dogs. Serum testosterone concentration decreased significantly by the onset of aspermia on study day 14 (four dogs) or day 21 (one dog), and was baseline on day 105 (week 15). In contrast, serum AMH started to increase only after the onset of aspermia and reached the maximum detectable concentration of the assay by day 49-105 in individual dogs. Testicular LHR gene expression in DES was lower than in CON and PRE (P < 0.0001), while AR gene expression in DES was similar to CON and significantly higher than PRE (P < 0.0001). Testicular AMH expression in DES was intermediate compared to the lowest mRNA levels found in CON and the highest in PRE (P ≤ 0.006). AMHR2 gene expression was similar between groups. AMH protein was detected in Sertoli cells only, while AMHR2 immunoreactivity was principally detected in Leydig cells which appeared to be increased in DES. INSL3 and RXFP2 gene expression was significantly downregulated in the DES testis along with noticeably weak Leydig cell immunosignals compared to CON. In conclusion, deslorelin treatment caused testicular LH insensitivity without affecting androgen sensitivity, and de-differentiation of Sertoli and Leydig cells. In DES, upregulation of the AMH-AMHR2 feed-back loop and downregulation of the INSL3-RXFP2 feed-forward loop are paracrine-autocrine mechanisms that may additionally regulate testosterone production independent of gonadotropins. Our results support AMH and INSL3 as unique biomarkers and paracrine-autocrine regulators of testis function involved in the intimate interplay between Sertoli and Leydig cells.

Practical experience with artificial insemination (AI) using fresh chilled and frozen semen in mares.

The objective of this study was to compare the efficiency of artificial insemination (AI) carried out with frozen and fresh, diluted and chilled semen under field conditions. One hundred and twenty-nine mares of different breeds were included in the study. Eighty-one out of the 107 mares inseminated with fresh, chilled semen got pregnant. Seven pregnant mares aborted and 74 foals were born. Out of the 22 mares inseminated with frozen semen, 17 mares got pregnant. Two mares out of the 17 pregnant mares aborted and finally 15 healthy foals were born. No difference was found between the two groups in the ratio of the foals born (P > 0.05). The comparison of medians for the number of insemination cycles did not show significant differences. However, a significant difference (Kruskal-Wallis test, P = 0.014) was found in the number of the inseminations per conception in favour of frozen semen (2.5 vs. 1.8 with fresh chilled and frozen semen, respectively). The Cox regression revealed that the type of semen has a significant impact (P < 0.001) on the service period (duration of the insemination period): the use of frozen semen prolonged the insemination period. This could be due to management issues, since re-insemination with frozen semen took place after only one/a few missed oestrous cycles not used for AI.

Possible effects of pituitary adenylate cyclase activating polypeptide (PACAP) on early embryo implantation marker HB-EGF in mouse.

Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated as a hypothalamic neuropeptide stimulating adenylate cyclase activity. Besides its neuroprotective effects, numerous data proved its role in reproductive processes. However, there are limited data on its role in preimplantation embryo development and implantation. Our aim was to analyse the mRNA expression of Adcyap1 (coding region of PACAP) and Hbegf [coding region of HB-EGF (Heparin-binding EGF-like growth factor)] in embryos and pregnant uterus to investigate the possible correlation between them. Eight-week-old BDF1 mice were superovulated and subsequently mated overnight or left in their cage after hCG treatment. Day4 embryos were flushed from mated females. After morphological analysis, Adcyap1 and Hbegf gene expression of embryos and uterine tissues was assessed with qPCR. Our results showed significantly higher Adcyap1 and Hbegf mRNA levels in females producing embryos compared to non-mated ones. Robust elevation of Adcyap1 and slight elevation of Hbegf were detected in females with blastocyst embryos compared with non-blastocysts. We found low rate of Hbegf mRNA expression in uncompacted embryos, whereas morulae and blastocysts expressed high amounts of Hbegf. However, we did not find detectable Adcyap1 mRNA in embryos. Strong correlation was found between uterine tissue and embryonic Hbegf levels, slight correlation between uterine Adcyap1 and Hbegf levels. Uterine tissue Adcyap1 and embryonic Hbegf showed no correlation. In summary, our present data show, for the first time, the correlation between PACAP and HB-EGF mRNA expression suggesting that PACAP might play a role during the peri-implantation period of early mouse embryo development.

Effects of T-2 and Fumonisin B1 combined treatment on in vitro mouse embryo development and blastocyst quality.

In the temperate region, most common mycotoxins are members of fusariotoxins. It often happens that food and forage are contaminated with two or more fusariotoxins at the same time. Effects of co-contamination are poorly documented, especially in the field of reproduction. The aim of our study was to assess the combined effect of the two common fusariotoxins, T-2 toxin (T-2) and Fumonisin B1 on early mouse embryo development in vitro. Embryo culture media contained either (1) 0.5 ng/ml T-2; 1, 2, or 10 ng/ml FB1 (group A, B, and C, respectively); or (2) 0.5 ng/ml T-2 and 1, 2, or 10 ng/ml FB1 (group TA, TB, and TC, respectively). Developmental rate, blastocoel expansion, cell number, and micronucleus rate were measured following 96 h culture. Although the developmental rate was similar to the control group (86.43% vs. 83.33, 78.79, 85.98, and 86.67%, respectively) in the case of single toxin treatments, the combined treatments induced significant decreases (14.5, 33.6, and 22.8% in TA, TB, and TC, respectively). The proportion of late blastocysts was lower in all treatments compared to control (83.6% vs. 0-83.6%). Combined treatment resulted in a significantly lower proportion of late blastocysts (25% in TA and 0% in TB and TC). Cell numbers decreased in all toxin-treated groups with a higher rate after combined treatments. No differences were detected in the micronucleus rate in the single or combined treatments compared to control. Our study shows that T-2 and FB1 toxins do not necessarily decrease the developmental rate, but co-contamination results in a significantly lower blastocyst rate and disturbs the blastocoel expansion as well. One possible explanation of this observation could be that the presence of two mycotoxins in the culture media intensifies their negative effects. All toxin treatments decreased the cell number in the blastocysts and this negative effect was more expressed after combined treatment.

T-2 mycotoxin slows down the development of mouse blastocysts, decreases their blastomere number and increases chromatin damage.

The mycotoxin T-2 has many harmful effects on mammalian cells and reproductive functions. In the present study, the in vitro effect of T-2 toxin on mouse blastocysts was examined. Embryos were cultured in media supplemented with 0.5, 0.75 and 1 ng/ml T-2. Different exposure times were applied [96 h (treatment I) or 24 h following 72 h in toxin-free media (treatment II)]. Blastomere number, nuclear chromatin status and blastocoel formation were investigated in blastocysts. Our data show that the effect of T-2 toxin may vary depending on the stage of the embryo at the start of exposure. At 96 h of exposure, the blastocysts had blastomeres with normal chromatin quality but their developmental potential was decreased. After 24 h of exposure applied following a 72-h culture, blastomeres had a higher level of chromatin damage, although their developmental potential was the same as in the control embryos. In both cases, decreased mitotic rate was found, which resulted in decreased blastomere number even at low toxin concentration.

The impact of laser-assisted hatching on the outcome of frozen human embryo transfer cycles.

Biochemical modifications of zona pellucida (ZP) result in zona hardening. Zona hardening (ZH) is induced by several factors such as advancing maternal age, in vitro culture conditions and cryopreservation and adversely effects implantation. The objective of the clinical study was to determine whether or not laser-assisted hatching (LAH) applied on day 3 frozen embryos improves the outcome of frozen embryo transfer (FET) cycles in patients with recurrent implantation failure and/or advanced female age. In total, 413 patients of different ages with recurrent implantation failure (maximum three cycles) were involved into the study. Patients were allocated randomly into LAH and control groups. On the day of FET, after thawing and just before FET, the ZP was thinned using a laser system. In the control group no treatment was applied on frozen embryo before transfer. The main outcome measures were clinical pregnancy rate. Overall, the results indicate a tendency that LAH increased (P = 0.08) clinical pregnancy. However, for patients older than 37 years, LAH increased pregnancy rates significantly (P = 0.03). In the LAH and control groups, the age of patients and the number of transferred embryos influenced pregnancy rates (P = 0.01). For patients older than 37 years, no effect of number of transferred embryos was detected (P = 0.14). The incidence of multiple pregnancies also increased in the LAH group (P = 0.01). In conclusion, in older woman, to overcome the negative effect of zona hardening, LAH could be performed on frozen embryos as a routine strategy before FET in frozen cycles in order to increase the possibility of pregnancy formation.

Effects of T-2 mycotoxin on in vitro development and chromatin status of mouse embryos in preimplantation stages.

T-2 toxin is a mycotoxin produced by phytopathogenic fungi of the Fusarium genus and has many well-studied deleterious effects on mammalian cells and reproductive tract. Despite the wide scale studies, the effects on preimplantation stage embryos are lacking. The aim of our study was to investigate the impact of T-2 on the cleavage stage of mouse embryos with regard to development to blastocysts and nuclear chromatin status.Six-weeks-old BDF1 female mice were superovulated and placed together overnight with mature males. Zygotes were flushed 20 h after human chorionic gonadotropin injection and divided randomly into treated (supplemented with 0.5, 0.75, and 1 ng/ml T-2) and nontreated (control) groups. Embryos were cultured in vitro for 96 h. Developmental stage was evaluated in the 72(nd)- and 96(th)-h for assessment of development dynamics. At the end of culture period, blastocysts from treated and control groups with normal morphology were selected for nuclear chromatin analysis. Blastocysts were categorized (grade A, B, and C) depending on the proportion of blasomeres with micronuclei and/or lobulated nuclei.Our data show significant decrease in the proportions of blastocysts in the 0.75 and 1 ng/ml toxin-supplemented groups compared with the control group. Blastocyst rate did not differ in embryos treated with 0.5 ng/ml T-2 but 24 h delay was found in blastocoel formation in all the treated groups. Only grade A (21.1%) and B (78.9%) blastocysts were found in low-toxin-contaminated group similar to the control ones (50-50%). Grade C embryos appeared in the 0.75 ng/ml (10%) treated group and the rate increased significantly (33.3%) in the highest contaminated group.T-2 mycotoxin has a harmful effect on early embryo development which results in decreased blastocyst proportion, delayed blastulation, and increased rate of chromatin damage.

Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification.

BACKGROUND: The ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts. METHODS: Mouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups. RESULTS: Cryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status. CONCLUSIONS: This study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

Cryopreservation of embryos and oocytes in human assisted reproduction.

Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.

Vitrification preserves chromatin integrity, bioenergy potential and oxidative parameters in mouse embryos.

BACKGROUND: The aim of this study was to evaluate the effects of vitrification on morpho-functional parameters (blastomere/chromatin integrity and bioenergy/oxidative potential) of mouse preimplantation embryos. METHODS: In vivo produced mouse (4/16-cell, morulae and blastocyst-stage) embryos were randomly divided into vitrification and control groups. For vitrification, embryos were exposed to a 2-step loading of ethylene glycol and propylene glycol, before being placed in a small nylon loop and submerged into liquid nitrogen. After warming, the cryoprotectants were diluted by a 3-step procedure. Embryo morphology, chromatin integrity and energy/oxidative status were compared between groups. RESULTS: Vitrification induced low grade blastomere cytofragmentation (P < 0.05) and low chromatin damage only in embryos at the morula stage (P < 0.001). Mitochondrial (mt) distribution pattern was affected by vitrification only in early embryos (P < 0.001). Mitochondrial activity did not change upon vitrification in morula-stage embryos but it was reduced in blastocyst-stage embryos (P < 0.05). Intracellular ROS levels significantly increased in embryos at the morula and blastocyst stages (P < 0.001). Colocalization of active mitochondria and ROS increased only in vitrified blastocysts. CONCLUSIONS: In conclusion, this study elucidates the developmentally-related and mild effects of vitrification on morphology, nuclear and bioenergy/oxidative parameters of mouse embryos and demonstrates that vitrification is a suitable method for preserving predictive parameters of embryo ability to induce a full-term pregnancy.