In vitro bovine embryo production in media modified with the addition of guaiazulene or ghrelin

The aim of this thesis was to study the effects of antioxidants during the phases of in vitro bovine embryo production, and to unravel their role on embryo yield and quality after quantification of particular gene markers. Under conditions that do not promote oxidation (low oxygen concentration) two antioxidants, one exogenous-the guaiazulene- and one endogenous –the ghrelin-were studied. Oocytes collected from slaughtered cows were in vitro matured for 24 hours in TCM199 supplemented with FCS and EGF. Matured oocytes were fertilized, the gametes were cocultured for 24 hours and putative zygotes were cultured in groupos of 25 in synthetic oviductal fluid. Day 7 blastocyst were frozen in liquid nitrogen for analysis of mRNA abundance of genes related to oxidation, metabolism, implantation, and apoptosis. Addition of 0.1mM or 0.01mM of guaiazulene in IVM or IVC media did not affect either blastocyst formation rate or the expression of the 7 genes studied. Modification of IVM medium with the addition of 200 pg/ml, 800 pg/ml and 2000pg/ml of ghrelin significantly suppressed blastocyst formation rates. When maturation time was shortened to 18 hours, more ghrelin treated oocytes reached metaphase II compared to untreated controls, no difference was noted after 24 hour IVM. Irrespectively of the duration of IVM, cumulus layer was more expanded in ghrelin treaterd oocytes compared to controls. 18 hour oocyte maturation in the presence of ghrelin, increased blastocyst yield compared to that of oocytes matured for 24 hours. Significant differences were detected in various genes’ expression giving an overall notiuon that oocytes matured for 24 hours in the presence of ghrelin are over matured or aged. Nonetheless, the hatching rate of blastocysts that were produced from oocyted exposed to ghrelin, were high suggesting improved embryo quality.The presence of ghrelin at the aforementioned concentrations, in the culture medium, significantly suppressed blastocyst formation rate. However, daily replacement of the culture medium reversed the former result causing a significant increase in blastocyst yield. Differences were detected in mRNA abundance of various genes revealing that embryos produced in the presence of ghrelin can be considered of improved quality. Our results provide evidence that ghrelin accelerates oocyte maturation, and drastically affects early embryo development. Collectively, the results of the present thesis show that oxidation is not a major hurdle during IVP, provided that the conditions used do not promote the development of oxidative phenomena. Conversely, endogenous antioxidant substances – such as ghrelin- can potently affect embryo yield and quality by interfering through multiple pathways related to developmental competence, metabolism, methylation, apoptosis and/or oxidation.Σκοπός της διατριβής ήταν να διερευνηθεί ο ρόλος αντιοξειδωτικών ουσιών σε όλα τα στάδια της εξωσωματικής παραγωγής εμβρύων βοοειδών, και να αποτυπωθεί ο ρόλος τους στην ποσοτική απόδοση σε έμβρυα και στην ποιότητα τους, με εκτίμηση συγκεκριμένων γονιδιακών δεικτών. Σε περιβάλλον που δεν προάγει την ανάπτυξη οξειδωτικών φαινομένων (χαμηλή συγκέντρωση οξυγόνου), μελετήθηκαν δύο αντιοξειδωτικές ουσίες, μια εξωγενής – το γουαϊαζουλένιο και μία ενδογενής , η γκρελίνη. Ωάρια σφαγίων αγελάδων ωρίμαζαν για 24 ώρες σε TCM199 εμπλουτισμένο με ορό εμβρύου μόσχου και επιδερμικό αυξητικό παράγοντα, στη συνέχεια γονιμοποιούνταν, οι γαμέτες συνεπωάζονταν για άλλες 24 ώρες και τέλος οι ζυγώτες καλλιεργούνταν σε ομάδες των 25 σε συνθετικό υγρό ωαγωγού. Βλαστοκύστεις της ημέρας 7 καταψύχονταν για ποσοτικό προσδιορισμό (qRT-PCR) του mRNA γονιδίων, που σχετίζονται με την οξείδωση, το μεταβολισμό, τη δυνατότητα εμφύτευσης του εμβρύου, την απόπτωση. Προσθήκη γουαϊαζουλένιου σε συγκεντρώσεις 0.1 mM και 0.01mM στην IVM ή στην IVC δεν επηρέασε το ποσοστό παραγωγής εμβρύων ούτε διαφοροποίησε την έκφραση 7 γονιδίων που μελετήθηκαν. Προσθήκη γκρελίνης σε συγκεντρώσεις• 200 pg/ml, 800 pg/ml και 2000pg/ml, στο υπόστρωμα ωρίμανσης μείωσε σημαντικά το ποσοστό παραγωγής βλαστοκύστεων. Με σύμπτυξη του χρόνου ωρίμανσης στις 18 ώρες βρέθηκε ότι σε σύγκριση με τους αντίστοιχους μάρτυρες, περισσότερα ωάρια που καλλιεργήθηκαν για 18 ώρες με την παρουσία γκρελίνης έφτασαν στην μετάφαση ΙΙ, ενώ δεν ανιχνεύθηκαν διαφορές για τις ομάδες στις οποίες η ωρίμανση διήρκεσε 24 ώρες. Στις 18 και 24 ώρες, η στιβάδα των κοκκωδών κυττάρων ήταν περισσότερο διατεταμένη στα COCs που ωρίμασαν υπό την παρουσία γκρελίνης. To ποσοστό σχηματισμού βλαστοκύστεων ήταν μεγαλύτερο μετά από ωρίμανση των ωαρίων για 18 ώρες, σε σχέση με αυτό των 24. Διαφορές εντοπίστηκαν σε διάφορες γονιδιακές εκφράσεις, οι οποίες οδηγούν στην εκτίμηση ότι η παρουσία της γκρελίνης για 24 ώρες οδηγεί στην υπερ-ωρίμανση / γήρανση των ωαρίων. Όμως, τα υψηλά ποσοστά εκκόλαψης των βλαστοκύστεων που αναπτύχθηκαν από ωάρια, τα οποία εκτέθηκαν σε γκρελίνη, δείχνουν ότι οι τελευταίες είναι καλύτερης ποιότητας από αυτές των ομάδων των μαρτύρων. Η παρουσία γκρελίνης στο υπόστρωμα καλλιέργειας των εμβρύων μείωσε σημαντικά το ποσοστό παραγωγής βλαστοκύστεων. Όμως η καθημερινή ανανέωση του υποστρώματος αύξησε σημαντικά το ποσοστό των παραγόμενων εμβρύων. Διαφορές ανιχνεύθηκαν στην έκφραση των γονιδίων, που οδηγούν στην εκτίμηση ότι η γκρελίνη βελτίωσε την ποιότητα των εμβρύων. Συνάγεται ότι η δράση της γκρελίνης, αφορά τόσο επιτάχυνση της διαδικασίας ωρίμανσης των ωαρίων, αλλά και σε επίπεδο ανάπτυξης του πρώιμου εμβρύου. Συμπερασματικά, τα αποτελέσματα της παρούσας διδακτορικής διατριβής δείχνουν ότι όταν οι συνθήκες παραγωγής δεν ευνοούν την οξείδωση, το οξειδωτικό στρες δεν είναι μείζον πρόβλημα για την εξωσωματική παραγωγή εμβρύων. Αντιθέτως, ενδογενείς αντιοξειδωτικές ουσίες –όπως η γκρελίνη- είναι δυνατό να επηρεάσουν την διαδικασία της παραγωγής και την ποιότητα των εμβρύων μέσω πολλαπλών λειτουργικών οδών, που σχετίζονται με το μεταβολισμό, τη δυνατότητα ανάπτυξης, την απόπτωση, τη μεθυλίωση ή και την οξείδωση

Association between physical properties of cervical mucus and ovulation rate in superovulated cows

Possible associations between certain physical properties of cervical mucus (CM) and ovulation rate were studied in 21 superovulated Holstein cows. In CM samples collected at the beginning of estrus (0 h) and in 4 h intervals for the following 24 h, the pH, the spinnbarkeit (spinability), and the crystallization value were measured. Blood samples, collected at the same time points with CM samples, were assessed for progesterone and estradiol concentrations. At 48 h the number of ovulated follicles was counted by transrectal ultrasonography and the animals were allotted into 2 groups according to the occurrence of at least one (group A, n = 16) or no (group B, n = 5) ovulations. The pH was lower (P < 0.05) at 8 h (7.00 + 0.24) in group A compared with group B (7.55 + 0.12). In group A, spinnbarkeit was significantly lower at 0 h and 20 h, and higher at 8 h and 16 h compared with group B (0 h: 2.50 + 0.82 versus 6.95 + 0.41; 20 h: 3.00 + 1.89 versus 5.38 + 0.94; 8 h: 7.00 + 0.87 versus 2.75 + 0.43; 16 h: 7.00 + 1.41 versus 4.30 + 0.71, for groups A versus B, respectively). Crystallization was significantly lower at 4 h (2.00 + 0.63) and 20 h (1.50 + 0.82) in group A compared with group B (3.13 + 0.32 at 4 h and 3.00 + 0.41 at 20 h). Progesterone at all time points, and estradiol at 16 h, 20 h, and 24 h were lower (P < 0.05) in group A than in group B. The pH, crystallization, estradiol, and progesterone differed (P < 0.05) within one group, while sbk differed within both groups

Composition, Organoleptic Characteristics, Fatty Acid Profile and Oxidative Status of Cow’s Milk and White Cheese after Dietary Partial Replacement of Soybean Meal with Flaxseed and Lupin

The effect of partial substitution of soybean meal by equal quantities of flaxseed and lupins in diets of Holstein dairy cows and heifers was investigated. A total of 6 animals (30 multiparous and 30 primiparous) were allocated into two equal groups in a randomised block design and fed control (group CO) or modified (group FL) TMR diets from three weeks prior to calving until day 40 postpartum. The TMR of group CO contained corn, barley, soybean meal, rapeseed cake, corn silage, and Lucerne hay, whereas in group FL equal quantities of whole flaxseed and lupins were used to replace 50% of the soybean meal in the TMR. All animals were fed twice daily with a daily allowance of 24 kg dry matter intake per animal. Milking was carried out three times daily and milk yield was recorded during every milking. Milk samples were analysed for chemical composition and SCC content. White cheeses were manufactured from bulk milk of each group at industrial level. Bulk milk and white cheese were analysed for chemical composition and fatty acid profile; cheese was also assessed for its organoleptic properties. Results indicate that milk yield did not differ among groups. Lipid oxidation values were similar among the groups, for both milk and cheese. However, FL inclusion resulted in lower (p p p p p p p p p < 0.05) when compared with that of group CO. The white cheese of cows fed diets with flaxseed and lupins showed compositional and organoleptic properties quite similar to control group cheese; aroma, texture, and color were acceptable and desirable in both cheeses. However, increased levels of n-3 polyunsaturated fatty acids were found in the cheese of FL fed animals. The substitution of soybean meal by flaxseed and lupins in diets of Holstein cows warrants further investigation, especially towards the production of cheese that meet the consumers’ demand for novel and healthier dairy products

Feeding Flaxseed and Lupins during the Transition Period in Dairy Cows: Effects on Production Performance, Fertility and Biochemical Blood Indices

Flaxseed and lupin seed were offered as an alternative dietary approach in dairy cows, through the partial substitution of soybean meal. Milk production and fertility traits were investigated. A total of 330 animals were allocated into two groups, treated (n = 176) and control (n = 154). From each group, 30 animals were selected for hematological and cytological studies. The experimental feeding period lasted for 81 days (25 days prepartum and 56 days postpartum). The control ration (group C) contained corn, barley, soybean meal, rapeseed cake, corn silage and lucerne hay; whereas, in the treatment group (group T), 50% of the soybean meal was replaced by an equal mixture of flaxseed and lupins. The two rations were formulated to be isonitrogenous and isoenergetic. Milk samples were analyzed for chemical composition, somatic cell count (SCC) content and total colony forming units (CFU). Blood samples were collected, and serum was analyzed for non-esterified fatty acids (NEFA), acute phase proteins (haptoglobin and serum amyloid) and lipid oxidation indices, namely thiobarbituric-acid-reactive substances (TBARS) and catalase activity. To assess polymorphonuclear neutrophils (PMN) numbers, endometrial samples from each cow were collected on days 21 and 42. No difference was recorded between groups in milk yield (p > 0.05). In multiparous cows, NEFA (mMol/L) concentrations were significantly lower in group T than in group C on day 14 (p > 0.009) and on day 42 (p = 0.05), while no difference was detected in the group of primiparous cows. At all time points, serum TBARS and catalase values were similar in both groups (p > 0.05). Multiparous cows in group T expressed the first postpartum estrus and conceived earlier than cows in group C (p ≤ 0.05). Between days 21 to 42 postpartum, the PMN reduction rate was higher in group T animals (p ≤ 0.05). Acute phase protein levels were in general lower in group T animals, and at specific time points differed significantly from group C (p ≤ 0.05). It was concluded that the partial replacement of soybean meal by flaxseed and lupins had no negative effect on milk yield or milk composition, and improved cow fertility; which, along with the lower cost of flaxseed and lupins mixture, may increase milk production profitability

Oviductal epithelial cells transcriptome and extracellular vesicles characterization during thermoneutral and heat stress conditions in dairy cows

12 Pág.This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH – CREATE – INNOVATE (Acronym: the summer cow, Project code: T1EDK-01078). Dimitrios Rizos was supported by the Spanish Ministry of Science and Innovation (PID2019-111641RB-I00), and Karina Cañon-Beltrán by Minciencias-Colombia Postdoctoral Fellowship (848–2019)Peer reviewe

The In Vitro Impact of the Herbicide Roundup on Human Sperm Motility and Sperm Mitochondria

Toxicants, such as herbicides, have been hypothesized to affect sperm parameters. The most common method of exposure to herbicides is through spraying or diet. The aim of the present study was to investigate the effect of direct exposure of sperm to 1 mg/L of the herbicide Roundup on sperm motility and mitochondrial integrity. Sperm samples from 66 healthy men who were seeking semen analysis were investigated after written informed consent was taken. Semen analysis was performed according to the World Health Organization guidelines (WHO, 2010). Mitochondrial integrity was assessed through mitochondrial staining using a mitochondria-specific dye, which is exclusively incorporated into functionally active mitochondria. A quantity of 1 mg/L of Roundup was found to exert a deleterious effect on sperm’s progressive motility, after 1 h of incubation (mean difference between treated and control samples = 11.2%) in comparison with the effect after three hours of incubation (mean difference = 6.33%, p &lt; 0.05), while the relative incorporation of the mitochondrial dye in mitochondria of the mid-piece region of Roundup-treated spermatozoa was significantly reduced compared to relative controls at the first hour of incubation, indicating mitochondrial dysfunction by Roundup. Our results indicate that the direct exposure of semen samples to the active constituent of the herbicide Roundup at the relatively low concentration of 1 mg/L has adverse effects on sperm motility, and this may be related to the observed reduction in mitochondrial staining

Effects of melatonin treatment on follicular development and oocyte quality in Chios ewes - Short communication

Follicular development and oocyte quality were assessed by laparoscopic observation and in vitro fertilisation, respectively, in melatonin-treated (Group M) and control (Group C) anoestrous Chios ewes (n = 10 in each group). Fourteen days after melatonin insertion, all ewes had laparoscopic evaluation of the follicular population followed by oocyte pick-up (OPU); on day 22 intravaginal progestagen sponges were inserted for 14 days. Two days after sponge removal the follicular population was re-evaluated and a second follicular aspiration was performed. Collected oocytes from the second OPU underwent in vitro maturation, fertilisation and culture. The number of large follicles was higher in Group M than in the control ewes during the first OPU and tended to be so (P = 0.06) at the second. Morphologically, oocytes collected from controls were of better quality than those from Group M; however, more oocytes collected from melatonintreated animals fertilised and developed in vitro . These results indicate that melatonin is a potent regulator of follicular development and oocyte competence during the anoestrous period of the ewe. © 2009 Akadémiai Kiadó