Heat Stress: A Serious Disruptor of the Reproductive Physiology of Dairy Cows.

Global warming is a significant threat to the sustainability and profitability of the dairy sector, not only in tropical or subtropical regions but also in temperate zones where extreme summer temperatures have become a new and challenging reality. Prolonged exposure of dairy cows to high temperatures compromises animal welfare, increases morbidity, and suppresses fertility, resulting in devastating economic losses for farmers. To counteract the deleterious effects of heat stress, cattl e employ various adaptive thermoregulatory mechanisms including molecular, endocrine, physiological, and behavioral responses. These adaptations involve the immediate secretion of heat shock proteins and cortisol, followed by a complex network of disrupted secretion of metabolic and reproductive hormones such as prolactin, ghrelin, ovarian steroid, and pituitary gonadotrophins. While the strategic heat stress mitigation measures can restore milk production through modifications of the microclimate and nutritional interventions, the summer fertility records remain at low levels compared to those of the thermoneutral periods of the year. This is because sustainment of high fertility is a multifaceted process that requires appropriate energy balance, undisrupted mode of various hormones secretion to sustain the maturation and fertilizing competence of the oocyte, the normal development of the early embryo and unhampered maternal-embryo crosstalk. In this review, we summarize the major molecular and endocrine responses to elevated temperatures in dairy cows, as well as the impacts on maturing oocytes and early embryos, and discuss the consequences that heat stress brings about in dairy cattle fertility.

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.

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 < 0.05) protein carbonyls and higher (p < 0.05) phenolic compounds in both milk and cheese samples. Milk from the FL group had decreased palmitic (p < 0.05) and myristic (p < 0.05) and increased oleic (p < 0.05) and linolenic acid (p < 0.05) when compared to group CO. White cheese from group FL showed a decrease in saturated fatty acids (SFA) (p < 0.05), an increase in monounsaturated fatty acids (MUFA) (p < 0.05), and a higher increase in polyunsaturated fatty acids (PUFA) (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.

Genetic diversity and thermotolerance in Holstein cows: Pathway analysis and marker development using whole-genome sequencing.

Heat stress causes extensive losses in the dairy sector, due to negative effects on milk production and reproduction. Cows have evolved a series of protective mechanisms, (physiological, biochemical, behavioural) to cope with the thermostressing environments, which have allowed the preservation of productive and reproductive potential of specific animals during summer; these animals are considered thermotolerant and could be used to design programs of selective breeding. These programs, targeting the generations of a population of heat-resistant animals, would increase the frequency of the desired phenotypes, tackling the financial losses on one hand and reducing the carbon footprints of the dairy sector on the other. The development of genomics techniques has enabled genome wide variant calling, to detect SNPs associated with the desired phenotypes. In this study, we used a comparative genomics approach to detect genetic variation associated with thermotolerance and to design molecular markers for characterizing the animals as tolerant/sensitive. A total of 40 cows from each group were split in four sequencing pools and a whole-genome sequencing approach was used. Results and conclusion: Genome-wide genetic variation between groups was characterized and enrichment analysis revealed specific pathways which participate in the adaptive mechanisms of thermotolerance, implicated into systemic and cellular responses, including the immune system functionality, Heat Stress and Unfolded Protein Response. The markers made a promising set of results, as specific SNPs in five genes encoding for Heat Shock Proteins were significantly associated with thermotolerance.

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

In this study, the transcriptome of oviductal epithelial cells and certain characteristics of their extracellular vesicles of dairy cows were described under thermoneutral and heat stress conditions. Twenty cows were compared in springtime at THI = 65.6 ± 0.90 and in summertime at THI = 78.36 ± 2.73. During each season, the estrous cycles of the cows were synchronized, and on day 3 of the ensuing cycle, a blood sample was collected for progesterone determination, while their oviducts were collected after slaughter. Epithelial cells and oviductal fluid were collected from the oviduct ipsilateral and contralateral to the corpus, respectively. For the gene expression study, a comparative transcriptomic approach, using RNASeq, was performed on cells collected from the ipsilateral and the contralateral oviducts. The size and the concentration of extracellular vesicles (EVs) at both seasons were analyzed using Transmission Electron Microscopy and Nanoparticle tracking analysis and specific proteins were detected by Western blotting. Progesterone concentration was higher during the thermoneutral period. Between seasons, divergent expression of genes related to immune system, contractility, gamete protection and lncRNAs was found. The size and the concentration of the EVs did not differ between seasons, however, the concentration in the ipsilateral oviduct tended to be lower (p = 0.09) from the contralateral one in the summer, but not in the spring. Our results show for the first time that HS could be involved with alterations in the oviductal cells' gene expression and in the changes in concentration of EVs in the oviductal lumen. Our results imply that the altered oviductal environment during HS could be associated with the suppressed summer fertility in dairy cows.

The Effects of Heat Shock Protein 70 Addition in the Culture Medium on the Development and Quality of In Vitro Produced Heat Shocked Bovine Embryos.

The aims of the present study were to examine the effects of HSP70 addition in the in vitro culture medium of day 3 embryos on their developmental competence and quality. Bovine oocytes (n = 1442) were in vitro matured, inseminated and cultured for the first two days according to standardized methods. The presumptive zygotes were randomly allocated in three experimental groups: Control, C (embryos cultured at 39 °C throughout the culture period), group C41 (temperature was raised to 41 °C from the 48th to 72nd h post insemination (p.i.) and then it returned at 39 °C for the remaining culture period), and group H41 (the temperature modification was the same as in C41 and during heat exposure, HSP70 was added in the culture medium). Cleavage and embryo yield were assessed 48 h p.i. and on days 7, 8, 9, respectively and gene expression in day 7 blastocysts was assessed by RT-PCR. Blastocyst yield was the highest in group C39; and higher in group H41 compared to group C41. From the gene expression analyses, altered expression of 11 genes was detected among groups. The analysis of the orchestrated patterns of gene expression differed between groups. The results of this study confirm the devastating effects of heat stress on embryo development and provide evidence that HSP70 addition at the critical stages can partly counterbalance, without neutralizing, the negative effects of the heat insult on embryos, acting mainly through mechanisms related to energy deployment.

Ultrasonographic findings of the corpus luteum and the gravid uterus during heat stress in dairy cattle.

The objectives of this study were to assess alterations in, echogenic appearance, size and blood flow in the corpus luteum, the placentomes and the blood flow in umbilical and uterine arteries that heat stress can cause in cooled pregnant dairy cows. Pregnant cows were allocated in two groups and the gravid uteri, along with the ipsilateral corpora lutea were examined during the winter (group W, n = 9) or the summer (group S, n = 10). The grey-scale ultrasound and colour flow imaging of the corpus luteum and placentome were performed. In addition, the umbilical and uterine artery diameters and haemodynamic parameters in the vessels were calculated. At the time of ultrasonographic examination, cortisol concentrations were higher, and progesterone levels tended to be lower in group S compared to group W. The grey-scale ultrasound evaluation of corpora lutea and placentomes was lower in group S compared to group W. The diameter of umbilical artery and the blood volume in the vessel were less in group S than in group W. We infer that heat stress affects foetal blood supply and possibly the structure of placentomes and corpora lutea, but it differently affects the blood flow characteristics in the umbilical and uterine arteries.

Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes.

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

Developmental competence of heat stressed oocytes from Holstein and Limousine cows matured in vitro.

The negative effects of heat stress on dairy cattle's fertility have been extensively studied, but the relevant knowledge for beef cattle is rather limited. The aims of this study were to investigate the effects of HS during in vitro maturation on the developmental potential of oocytes derived from Limousine and Holstein cows and to estimate the effect of the differential gene expression of important genes in oocytes, cumulus cells and blastocysts in the growth competence between the breeds. In seven replicates, cumulus oocyte complexes from Holstein and Limousine cows were matured for 24 hr at 39°C (controls C; Hol_39, Lim_39) or at 41°C from hour 2 to hour 8 of IVM (treated T; Hol_41, Lim_41), fertilized, and presumptive zygotes were cultured for 9 days at 39°C. Cleavage and embryo formation rates were evaluated 48 hr post-insemination and on days 7, 8 and 9, respectively. From all groups, subsets of cumulus cells, oocytes and blastocysts were analysed for the relative expression of genes related to metabolism, stress, apoptosis and placentation. No difference was detected in cleavage rate or in blastocyst formation rate among the control groups. In both breeds, heat stress reduced blastocyst yield, but at all days the suppression was higher in Limousines. In Holsteins, altered gene expression was detected in cumulus cells (G6PD, GLUT1) and blastocysts (PLAC8), while in Limousines, differences were found in oocytes (G6PD, HSP90AA1), in cumulus cells (CPT1B, HSP90AA1, SOD2) and blastocysts (DNMT, HSP90AA1, SOD2). It appears that Holstein COCs are more tolerant than Limousine COCs, possibly due to compulsory, production driven selection.

Early embryo losses, progesterone and pregnancy associated glycoproteins levels during summer heat stress in dairy cows.

Objectives of this study were to characterize the effects of heat stress on pregnancy associated glycoproteins (PAG) and progesterone and its involvement in embryo survival. In trial 1, blood samples collected from days 29 to 36 post insemination were examined for the comparison of PAG concentrations between winter (n = 3721) and summer (n = 2388). In trial 2, embryo losses were assessed in winter (n = 144) and in summer (n = 133), in days 31 or 32 of pregnancy. Pregnancy diagnosis was carried out by ultrasonography on days 24 or 25, and it was repeated a week later; in the second occasion PAG concentration was also determined. In trial 3 the PAG and progesterone concentrations were assessed in days 33 to 36 in winter and summer. In trial 1 PAG levels did not differ between winter and summer, the conception rate and the proportion of uncertain pregnancies were higher in winter than summer. The likelihood of pregnancy was 10 to 15% higher in winter. In trial 2, the embryo death rate was higher in summer, but the PAG levels of cows that had embryo loss in summer were higher than those in winter. In both seasons, lower PAG levels were associated with higher risk of pregnancy loss, while embryo death was five times more likely to occur in summer than in winter and lower PAG concentrations were positively associated with higher risk of embryo loss. In trial 3, mean PAG levels were higher and of progesterone were lower during the summer than during the winter. We infer that despite the devastating effects of heat stress on cows' fertility, those early embryos that survive under continuous heat stress can form a well-functioning placenta; hence, the high embryo mortality rate observed during the summer months could be mainly attributed to luteal insufficiency.

A study on stress response and fertility parameters in phenotypically thermotolerant and thermosensitive dairy cows during summer heat stress.

It is well documented that heat stress (HS) causes subfertility in dairy cows. However, during the last ten years we have been observing that, under high temperature-humidity index (THI ≥ 75), despite the overall reduced fertility, some cows conceive at the first artificial insemination (AI). Here, we examined distinctive features of cows with conserved fertility under severe HS. From the databases of three herds, 167 lactating Holstein cows were selected; group TT cows (n = 57) conceived in the previous summer (THI ≥ 75) at the 1st AI, and group TS (n = 110) failed to conceive at the same period after at least 2 consecutive AIs. The animals calved in spring, and in August, blood samples were collected during a hot day (THI ≥ 81) for the determination of cortisol and HSP70 concentrations. In one farm, the validity of fertility data of the previous year was re-examined. In 28 cows from group TT and in 39 cows from group TS, the conception rate was examined during July and August. In 6 cows from each group (TT and TS) the oestrous cycles were synchronized, ovulation was induced with GnRH (THI = 80), and the concentration of the pre-ovulatory LH surge was determined in 9 blood samples. The progesterone concentration in the ensuing cycle was determined in blood samples collected every other day. Overall, cortisol and HSP70 were significantly lower in TT group compared to TS. More (p < .05) animals from group TT conceived at the first AI compared with those from group TS. The induced pre-ovulatory LH surge peaked at higher level (p < .002) in group TT than in group TS, while no difference was recorded among groups either in mean progesterone concentrations or in the duration of the ensuing oestrous cycle. These results are highly suggestive that thermotolerance in some dairy cows is an inherent characteristic, warranting further genetic investigation.

Short term temperature elevation during IVM affects embryo yield and alters gene expression pattern in oocytes, cumulus cells and blastocysts in cattle.

Heat stress causes subfertility in cattle by inducing alterations in steroidogenic capacity, follicular function and ovulation defects, which eventually negatively affect oocyte quality and embryo survival. Here, the effects of short, moderate temperature elevation during IVM, on embryo yield, and on the expression of various genes was evaluated. In 8 replicates, cumulus oocyte complexes (COCs) were matured for 24 h at 39 °C (controls n = 605) or at 41 °C from hour 2 to hour 8 of IVM (treated, n = 912), fertilized, and presumptive zygotes were cultured for 9 days at 39 °C. Cleavage and embryo formation rates were evaluated 48 h post insemination and on days 7, 8, 9 respectively. Cumulus cells, oocytes and blastocysts from 5 replicates were snap frozen for the relative expression analysis of genes related to metabolism, thermal and oxidative stress response, apoptosis, and placentation. In treated group, cleavage and embryo formation rates were statistically significantly lower compared with the control (cleavage 86.7% vs 74.2%; blastocysts: day 7, 29.9% vs 19.7%, day 8, 34.2% vs 22.9% and day 9 35.9% vs 24.5%). Relative mRNA abundance of three genes in cumulus cells (HSP90AA1, CPT1B, G6PD) and three genes in blastocysts (DNMT3A, PLAC8, GPX1) indicated significantly different expression between groups (p < 0.05)., The expression of G6PD, SOD2, GXP1 in oocytes and PTGS2 in blastocysts tended to differ among groups (0.05

Pancreatic anti-GP2 and anti-Saccharomyces cerevisiae antibodies in ruminants with paratuberculosis: A better understanding of the immunopathogenesis of Crohn's disease.

INTRODUCTION: Ruminants (cattle and sheep) with Mycobacterium avium (MAP)-induced paratuberculosis (ptb), the ruminant model of Crohn's disease (CD), exhibit pancreatic specific autoantibodies (PAB) against GP2 but not against CUZD1. Since anti-Saccharomyces cerevisiae antibodies (ASCAs) is a CD marker, we tested MAP-infected ptb ruminants for ASCA, and compared them with ruminants lacking evidence of anti-MAP serology or with ruminants, which were positive for anti-GP2 antibodies. MATERIAL AND METHODS: A total of 98 samples from ruminants (48 cattle and 50 sheep) were studied. IgG anti-MAP antibodies, and CD-related ASCA and anti-GP2 antibodies were tested by modified ELISAs. RESULTS: Nine cattle (18.75%) and 20 sheep (40%) were suffered from ptb. ASCA antibodies were present in 21/48 (43.7%) cattle and 10/50 (20%) sheep while anti-GP2 antibodies were present in 14/48 (29.2%) cattle, and 8/50 (16%) sheep. ASCA antibodies were more prevalent in anti-MAP antibody positive (14/29, 48.3%) than in anti-MAP negative ruminants (17/69, 24.6%, P=0.022) and also in anti-GP2 antibody positive (13/23, 56.5%) than in anti-GP2 negative ruminants (18/75, 24%, P=0.003). No association between ASCA and anti-MAP antibody concentrations were found (r=0.159, P=0.117). A significant association between ASCA and anti-GP2 antibody concentration were observed (r=0.211 and P=0.037). CONCLUSION: ASCA are present in a significant proportion of ruminants with ptb and correlate with anti-GP2 antibody positivity, a finding further supporting the notion that Crohn's disease and ptb share common immunological mechanisms of antigen-driven loss of self-tolerance.

A study on ghrelin and LH secretion after short fasting and on ghrelin levels at perioestrual period in dairy cattle.

In two experiments, we studied (a) the changes of LH secretion in heifers under different feeding schedules and (b) total ghrelin concentration at oestrus in cows and heifers. In experiment one, synchronized heifers were allocated in three groups (R, regularly fed controls; F, fasted; and F-F fasted-fed). One day after the completion of the oestrous induction protocol, group F and F-F animals stayed without feed for 24 hr; thereafter, feed was provided to R and F-F cattle; 2 hr later, GnRH was administered to all animals. Blood samples were collected for ghrelin, progesterone, LH and cortisol concentrations. Fasting caused increased ghrelin concentrations in groups F and F-F, while in response to GnRH, LH surge was significantly attenuated in groups F and F-F compared to R. In experiment 2, lactating cows and heifers were used. On day 9 of a synchronized cycle, PGF2α was administered, and blood samples were collected twice daily until the third day after oestrus and analysed for progesterone, estradiol, ghrelin, glucose and BHBA concentrations. No difference was recorded between groups in steroids and BHBA concentrations. In comparison to mid-luteal values, ghrelin concentrations significantly increased at perioestrual period in cows, but not in heifers. This study provides evidence that starving-induced elevated ghrelin concentrations can have suppressing effect on LH secretion, even after ghrelin's restoration to basal values and that during oestrus, ghrelin secretion is differently regulated in cows and heifers, likely being independent from oestradiol concentrations. Further research is required to identify the determining factors that drive the different regulation of ghrelin secretion in cows and heifers.

Effects of pregnancy and short-lasting acute feed restriction on total ghrelin concentration and metabolic parameters in dairy cattle.

The aims of this study were: to compare total ghrelin concentration throughout pregnancy between lactating cows and heifers, and to study the response to acute feed restriction in pregnant or non-pregnant heifers. Blood samples were collected each month of pregnancy from cows (n = 5) and heifers (n = 5) and analyzed for total ghrelin concentration. Compared to pre-conception values, ghrelin concentrations tended to be greater during 3rd month of pregnancy in heifers, whereas they were higher in the 7th, 8th and 9th months in lactating cows, but no difference was detected between lactating cows and heifers. In experiment two, pregnant (n = 4) and non-pregnant (n = 4) heifers were fasted for 24 h. Blood samples were collected 0, 4, 8, 12, 16 and 24 h of fasting and were assayed for, insulin, glucose, cortisol, BHBA and NEFA concentrations, and at time points 0, 8, 16 and 24 for total ghrelin determination. Compared to satiety, ghrelin concentrations were higher at 8th, 16th and 24th hour of fasting in pregnant and at 8th hour in non-pregnant animals, but no difference was detected between pregnant and non-pregnant heifers. Pregnant heifers had lower glucose concentrations than non-pregnant ones. Insulin concentrations were reduced at 4 and 8 h of fasting in pregnant heifers, and stayed unaffected in non-pregnant ones. Cortisol concentrations increased after 4th hour and remained elevated throughout the sampling period in pregnant heifers, while they increased at 24th h in non-pregnant animals. Here, we provide evidence that total ghrelin concentrations rise in response to feed restriction. Albeit no group effect was evident, our results imply that a) during the last trimester of pregnancy total ghrelin is secreted in different pattern between lactating cows and heifers, b) pregnant animals are more responsive than non-pregnant ones to hunger induced stress.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes.

The purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus-oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml-1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

Urokinase-type plasminogen activator does not affect in vitro bovine embryo development and quality.

The effects of modification of the in vitro embryo culture media (IVC) with the addition of urokinase-type plasminogen activator (u-PA) on the yield and/or quality of bovine embryos were examined in two experiments. In Experiment 1, denuded embryos were cultured in semi-defined synthetic oviductal fluid (SOF) for seven days, while in Experiment 2 embryos were co-cultured with cumulus cell monolayer in a serum-containing SOF medium. Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were determined in all spent IVC media. At the activity used (5 IU/ml), u-PA had no effect either on in vitro embryo production rates or on embryo quality as revealed by gene expression analysis of 10 important mRNA transcripts related to apoptosis, oxidation, implantation and metabolism. PAA and PAI analysis indicated the need for wellbalanced plasminogen activators and inhibitors as a culture environment for embryo development. However, more research is needed to unveil the mechanism by which u-PA is involved in in vitro embryo production systems.

Crohn's disease-specific anti-CUZD1 pancreatic antibodies are absent in ruminants with paratuberculosis.

BACKGROUND: Pancreatic autoantibodies (PABs) specifically recognizing GP2 and/or CUZD1 are present in more than 35% of patients with Crohn's disease (CrD). We have recently provided evidence of the presence of GP2-specific PABs in ruminants with paratuberculosis (ptb), a Mycobacterium avium paratuberculosis (MAP)-induced disease resembling CrD. OBJECTIVE: To assess whether anti-CUZD1 antibodies are also present in ruminants with ptb. METHODS: A total of 110 samples (73 cattle/37 sheep) were studied including 40 with ptb (24 cattle/16 sheep; 20 anti-GP2 antibody pos) and 70 without ptb (49 cattle/21 sheep; 10 anti-GP2 antibody pos). The samples were pre-characterized for anti-MAP and anti-GP2 antibodies by ELISA. Evidence of MAP was confirmed by PCR. Anti-CUZD1 antibody testing was performed by indirect immunofluorescence (IIF) based on transfected HEK293 cells expressing CUZD1. Anti-sheep or anti-cattle specific antisera were used as revealing antibodies. RESULTS: None of the ruminant sera had anti-CUZD1 antibodies by IIF testing at dilutions varying from 1/10 to 1/160. Methodological flaws were prevented by a series of tests. Control sera from anti-CUZD1 positive CrD samples have shown anti-CUZD1 antibody reactivity at various concentrations. Antibody reactivity to GP2-expressing HEK293 cells has confirmed the reactivity to GP2 in ruminant sera found positive for anti-GP2 antibodies by ELISA. CONCLUSION: The present study has found no evidence of anti-CUZD1 PABs in MAP-induced ptb. Our findings indicate that the induction of CUZD1-specific PABs is unrelated to MAP infection and that the mechanisms responsible for the loss of tolerance to GP2 and CUZD1 are probably quite distinct.

Relationship between electrical resistance of cervical mucus and ovarian steroid concentration at the time of artificial insemination in ewes.

The purpose of this study was to investigate whether fertile or non-fertile inseminations (AI) in synchronized ewes are correlated with the electrical resistance of cervical mucus (ERCM) and the ovarian steroid concentration. AIs were performed either at fixed-time (group A) or after estrus detection (group B). Retrospective analysis revealed that at AI, pregnant ewes had lower ERCM values and progesterone concentrations than non-pregnant ones (p<0.05). It appears that ERCM may be used as an additional index for fertility enhancement of inseminated ewes.

Daily supplementation with ghrelin improves in vitro bovine blastocysts formation rate and alters gene expression related to embryo quality.

Ghrelin is a gastric peptide having regulatory role in the reproductive system functionality, acting mainly at central level. Because the expression of ghrelin system (ghrelin and its receptor) has been detected in the bovine ovary, the objectives of the present study were to investigate whether ghrelin can affect the developmental potential of in vitro-produced embryos, and to test their quality in terms of relative abundance of various genes related to metabolism, apoptosis and oxidation. In the first experiment, in vitro-produced zygotes were cultured in the absence (control [C]) and in the presence of three concentrations of acylated ghrelin (200 pg/mL [Ghr200], 800 pg/mL [Ghr800]; and 2000 pg/mL [Ghr2000]); blastocyst formation rates were examined on Days 7, 8, and 9. In the second experiment, only the 800 pg/mL dose of ghrelin was used. Zygotes were produced as in experiment 1 and 24 hours post insemination they were divided into 4 groups; in two groups (C; without ghrelin; Ghr800 with ghrelin), embryos were cultured without medium replacement; in the remaining two groups (Control N and GhrN), the culture medium was daily renewed. A pool of Day-7 blastocysts were snap frozen for relative mRNA abundance of various genes related to metabolism, oxidation, implantation, and apoptosis. In experiment 3, embryos were produced as in experiment 2, but in the absence of serum (semi-defined culture medium). In experiment 1, no differences were detected between C, Ghr200, and Ghr2000, although fewer blastocysts were produced in group Ghr800 compared with C. In experiment 2, the lowest blastocysts yield was found in Ghr800, whereas daily renewal of ghrelin (Ghr800N) resulted to increased blastocysts formation rate, which on Day 7 was the highest among groups (P < 0.05). In experiment 3, ghrelin significantly suppressed blastocysts yield. Significant differences were detected in various relative mRNA abundance, giving an overall final notion that embryos produced in the presence of ghrelin were of better quality than controls. Our results imply a specific role of ghrelin in early embryonic development; however, the specific mode of its action needs further investigation.