Effect of season on the in vitro fertilizing ability of frozen-thawed Spanish bovine spermatozoa.

The purpose of the present study was to evaluate the effects of season on the in vitro fertilizing ability of bovine spermatozoa and subsequent embryo development. Bovine oocytes were matured and fertilized in vitro with Holstein dairy bull sperm cells collected and frozen in different seasons (winter, spring, and summer). On d 2 and 8 postinsemination, cleavage and blastocyst rates, respectively, were recorded; the blastocysts were graded for morphology. The number of sperm cells binding to the zona pellucida of oocytes, together with the number of nuclei in the developing blastocysts, were assessed after staining with Hoechst. No significant differences were observed among seasons in cleavage and embryo development rate. However, the proportion of "advanced blastocysts" was significantly higher in spring compared with winter and summer, with a corresponding decrease in the proportion of early blastocysts in spring compared with winter and summer. The number of sperm cells binding per oocyte was significantly lower in the oocytes inseminated with sperm samples collected in summer compared with winter or spring. Moreover, a significant interaction was observed in the number of sperm cells binding per oocyte between bull and season. Although no significant differences were observed among seasons in the number of nuclei per blastocyst, a significant interaction was observed between bull and season for this variable. Embryo development rate in in vitro fertilization appeared to be affected by season of semen collection, with sperm samples collected in spring being associated with a higher proportion of advanced blastocysts and better morphology than those collected at other times of the year.

Relationships between climate and sperm quality in dairy bull semen: A retrospective analysis.

The aim of the present study was to make a retrospective analysis of the relationship between climatic factors and sperm quality of frozen-thawed semen from bulls kept in temperate climates. Semen samples from 21 European dairy bulls from 2 countries were collected and cryopreserved in winter, spring, and summer. Sperm quality parameters such as kinematics, morphology, plasma membrane integrity, mitochondrial membrane potential, sperm chromatin structure assay, and reactive oxygen species were analyzed and correlated retrospectively with climate factors recorded by the local meteorological office. This study demonstrated that sperm quality parameters are more likely to be correlated with climate factors 1 or 2 mo before semen collection than in the month of semen collection. During the month of sperm collection, sperm kinematics, DNA fragmentation, and hydrogen peroxide production were the only sperm quality parameters related to climate factors, whereas 1 and 2 mo before sperm collection, normal morphology and additional sperm kinematics, in addition to DNA fragmentation and hydrogen peroxide production, were correlated with climate factors. In conclusion, dairy bull sperm quality is affected by climatic conditions, even in so-called temperate zones. The timing of heat stress during spermatogenesis determines which aspects of sperm quality are likely to be affected. Husbandry conditions for bulls used for semen collection should be adapted to allow the animals' physiological responses for temperature regulation within the scrotum to operate fully, to mitigate the effects of increased temperature and humidity. Extremes of temperature should be avoided.

Variation in scale cortisol concentrations of a wild freshwater fish: Habitat quality or seasonal influences?

A significant body of literature suggests that aquatic pollutants can interfere with the physiological function of the fish hypothalamic-pituitary-interrenal (HPI) axis, and eventually impair the ability to cope with subsequent stressors. For this reason, development of accurate techniques to assess fish stress responses have become of growing interest. Fish scales have been recently recognized as a biomaterial that accumulates cortisol, hence it can be potentially used to assess chronic stress in laboratory conditions. We, therefore, aimed to evaluate the applicability of this novel method for cortisol assessment in fish within their natural environment. Catalan chub (Squalius laietanus) were sampled from two sites; a highly polluted and a less polluted (reference) site, in order to examine if habitat quality could potentially influence the cortisol deposition in scales. We also evaluated the seasonal variation in scale cortisol levels by sampling fish at three different time points during spring-summer 2014. In each sampling, blood was collected to complement the information provided by the scales. Our results demonstrated that blood and scale cortisol levels from individuals inhabiting the reference site were significantly correlated, therefore increasing the applicability of the method as a sensitive-individual measure of fish HPI axis activity, at least in non-polluted habitats. Since different environmental conditions could potentially alter the usefulness of the technique, results highlight that further validation is required to better interpret hormone fluctuations in fish scales. Scale cortisol concentrations were unaffected by habitat quality although fish from the polluted environment presented lower circulating cortisol levels. We detected a seasonal increase in scale cortisol values concurring with an energetically costly period for the species, supporting the idea that the analysis of cortisol in scales reveals changes in the HPI axis activity. Taken together, the present study suggests that cortisol levels in scales are more likely to be influenced by mid-term, intense energetically demanding periods rather than by long-term stressors. Measurement of cortisol in fish scales can open the possibility to study novel spatio-temporal contexts of interest, yet further research is required to better understand its biological relevance.

Hair cortisol and progesterone detection in dairy cattle: interrelation with physiological status and milk production.

Hair cortisol concentrations (HCCs) and hair progesterone concentrations (HPCs) allow monitoring long-term retrospective steroid levels. However, there are still gaps in the knowledge of the mechanisms of steroid deposition in hair and its potential application in dairy cattle research. This study aimed to evaluate the potential uses of hair steroid determinations by studying the interrelations between HCC, HPC, physiological data from cows, and their milk production and quality. Cortisol and progesterone concentrations were analyzed in hair from 101 milking Holstein Friesian cows in a commercial farm. Physiological data were obtained from the 60 d prior to hair collection. Moreover, productive data from the month when hair was collected and the previous one were also obtained as well as at 124 d after hair sampling. Significant but weak correlations were found between HCC and HPC (r = 0.25, P < 0.0001) and between HPC and age (r = 0.06, P = 0.0133). High HCC were associated with low milk yields from the 2 previous months to hair sampling (P = 0.0396) and during the whole lactation (P < 0.0001). High HCC were also related to high somatic cell count (P = 0.0241). No effect of HCC on fat or protein content was detected. No significant correlations were detected between hair steroid concentrations and pregnancy status, days of gestation, parturition category (primiparous vs multiparous), number of lactations or days in milk. The relationship between physiological variables and HCC or HPC could depend on the duration of the time period over which hair accumulates hormones. Steroid concentrations in hair present high variability between individuals but are a potential tool for dairy cattle welfare and production research by providing a useful and practical tool for long-term steroid monitoring.

Analysis of ZP1 gene reveals differences in zona pellucida composition in carnivores.

The zona pellucida (ZP) is an extracellular envelope that surrounds mammalian oocytes. This coat participates in the interaction between gametes, induction of the acrosome reaction, block of polyspermy and protection of the oviductal embryo. Previous studies suggested that carnivore ZP was formed by three glycoproteins (ZP2, ZP3 and ZP4), with ZP1 being a pseudogene. However, a recent study in the cat found that all four proteins were expressed. In the present study, in silico and molecular analyses were performed in several carnivores to clarify the ZP composition in this order of mammals. The in silico analysis demonstrated the presence of the ZP1 gene in five carnivores: cheetah, panda, polar bear, tiger and walrus, whereas in the Antarctic fur seal and the Weddell seal there was evidence of pseudogenisation. Molecular analysis showed the presence of four ZP transcripts in ferret ovaries (ZP1, ZP2, ZP3 and ZP4) and three in fox ovaries (ZP2, ZP3 and ZP4). Analysis of the fox ZP1 gene showed the presence of a stop codon. The results strongly suggest that all four ZP genes are expressed in most carnivores, whereas ZP1 pseudogenisation seems to have independently affected three families (Canidae, Otariidae and Phocidae) of the carnivore tree.

Coenzyme Q10 supplementation during in vitro maturation of bovine oocytes (Bos taurus) helps to preserve oocyte integrity after vitrification.

Oocyte vitrification causes less cell stress than slow cooling, but cytoskeletal and spindle alterations may occur affecting the oocyte competence. In vitro maturation (IVM) supplementation with different antioxidant molecules has been performed to attenuate this harmful stress. Coenzyme Q10 (CoQ10 ) supplementation has previously shown to have positive effects in bovine and mouse in vitro embryo development. The aim of this study was to evaluate the effects of CoQ10 during bovine oocyte IVM and vitrification. Cumulus-oocyte complexes (COCs) (n = 311) were cultured under standard maturation conditions with 0 µM (control), 25 µM and 50 µM CoQ10 supplementation. After 22 hr, a cohort of 170 oocytes both from the control and from CoQ10 -supplemented groups were vitrified, warmed and returned to incubation until 24 hr of maturation, while the rest of the oocytes (n = 141) remained fresh. Then, oocyte survival was assessed morphologically by stereomicroscopy. Oocytes from all groups were then fixed and stained for assessing cortical granules (CG) migration and nuclear stage. High rates of oocyte MII progression and appropriate CG migration as a continuous layer beneath the plasma membrane were obtained both in control and in CoQ10 groups. Results showed that although vitrification has great impact in survival of IVM bovine oocytes, 50 µM CoQ10 supplementation significantly improved oocyte survival (p = .045) and reduced the premature CG exocytosis, helping to preserve the CG migration pattern (31.3% control vs. 54.5% in 50 µM CoQ10 ; p = .039), attenuating the negative effects of vitrification.

Effect of heat stress during in vitro maturation on developmental competence of vitrified bovine oocytes.

Heat-shock protein 70 (HSP70) plays a crucial role as intracellular cytoprotectant and molecular chaperone. A phenomenon of heat stress (HS) leads to production of these proteins that could be beneficial to cells during cryopreservation, which is also a stressful process for the cell. This study aimed to evaluate the protective effect of exposure of bovine oocytes to moderate HS during in vitro maturation (IVM) prior vitrification. First, oocytes were subjected to HS (41.5°C for 1 hr) at 0, 4, 8, 12 or 16 of IVM. Oocytes in vitro matured for 20 hr served as control group. Presence of HSP70 was detected at 20 hr by immunofluorescence. HSP70 expression was significantly higher when oocytes were subjected to HS at 8 hr of IVM. Next, oocytes were distributed into four groups: Control: IVM oocytes; VIT: oocytes vitrified/warmed at 20 hr of IVM; HS: oocytes subjected to HS at 8 hr of IVM; HS-VIT: oocytes subjected to HS at 8 hr of IVM and vitrified/warmed at 20 hr of IVM. Oocytes were fertilized at 24 hr of IVM, and cleavage and blastocyst yield were assessed. No significant differences were observed among treatments when cleavage rate was evaluated. However, fresh control and HS oocytes resulted in a significantly higher (p < .05) blastocyst rate when compared to VIT and HS-VIT groups, although no significant differences within fresh or vitrified groups were observed. In conclusion, HS did not have a negative impact on the oocyte competence but HS applied before vitrification, offered no benefits for embryo development.

Increased sperm cell production in ageing roosters by an oral treatment with an aromatase inhibitor and a natural herbal extract designed for improving fertility.

Rooster fertility peaks between 30 and 40 weeks of age and declines rapidly from 50 weeks of age. This is linked to a reduction in the number of spermatozoa in the ejaculate due to high density of spermatids in the seminiferous tubules of low-fertility roosters. In this study, we assessed the effects over spermatogenesis of both letrozole, an aromatase inhibitor, and a commercial herbal extract designed for improving fertility composed by Tribulus, Cinnamomum, Zingiber and Sativus. Forty-two-week-old Ross 308 roosters (n = 24) were distributed into four groups: control, letrozole (0.03 mg/kg), herbal extract (0.04 ml/kg) and letrozole+herbal extract. After 14 weeks of daily oral supplementation, their testes and epididymides were weighed, fixed and sectioned for assessment of spermatogenesis and quantification of sperm cells inside the lumen. Differences in seminiferous tubules measurements and density of sperm cells were tested using R software (version 3.0.1). Although body weight was not affected by the treatment, testes from animals treated with the combination of letrozole and herbal extracts were heavier than those from control animals. Animals treated with either letrozole or herbal extract, or their combination, showed a significant higher number of sperm cells inside the seminiferous tubules and epididymis than control animals (p < .05). These data suggest that the use of letrozole and the herbal extract could improve sperm cell production in ageing roosters. Future studies are needed to disclose the causal mechanisms involved and its effect on fertility and ejaculate features.

Effects of season on bull sperm quality in thawed samples in northern Spain.

The aim of the present study was to evaluate the possible effects of climate factors on sperm quality of Holstein dairy bulls housed in northern Spain. Semen samples from 11 Holstein dairy bulls were collected and cryopreserved in winter, spring and summer. Sperm quality parameters such as motility, morphology, plasma membrane integrity, acrosome status, mitochondrial membrane potential, DNA fragmentation index and reactive oxygen species were assessed. Samples collected in spring showed higher mean values of total and progressive motility compared with samples collected in winter. Mean values of average path velocity and straight-line velocity were higher in spring than in summer. The proportion of viable spermatozoa was higher in spring than in winter as was the proportion of viable spermatozoa with non-reacted acrosome. The proportion of live cells that were not producing superoxide or hydrogen peroxide was higher in samples collected in spring than in winter. No differences were found in sperm morphology or the DNA fragmentation index among seasons. In conclusion, results suggest that sperm quality of bulls housed in northern Spain is affected by season. Samples collected in spring appear to have better sperm quality than samples collected in other seasons.

Acute ACTH-induced elevations of circulating cortisol do not affect hair cortisol concentrations in calves.

In recent times, the detection of cortisol in hair is intended to be used as an animal-based indicator for the assessment of chronic stress. However, the relationship between the ability to report average values of long-term circulating cortisol concentrations and the sensitivity to acute peaks of cortisol is still unclear. To gain insight into this relationship, 24 Holstein-Friesian bull calves under the same management conditions were used in this study. Two injections of ACTH (at D0 and D7) were administrated to twelve animals with the aim to create two acute increases of serum cortisol concentrations. Blood samples were taken in order to determine the duration of serum cortisol peaks and to confirm a mediated response by the administration of ACTH. Cortisol concentrations from 14-day-old white hair samples collected from forehead and hip were analysed separately by EIA and compared with those from the control group. Serum cortisol analyses revealed an acute increase of cortisol concentrations for approximately three hours after each ACTH administration. Concentrations of hair cortisol from forehead and hip locations showed no differences between ACTH-administrated and control animals. Hair cortisol concentrations from 14-day old samples were not altered by two acute elevations of serum cortisol suggesting that hair cortisol is not masked by short and non-recurrent moments of stress. These results are a step forward in the validation of hair cortisol detection as a robust integrative measure of serum cortisol concentrations from an extended period of time.

Daily exposure to summer temperatures affects the motile subpopulation structure of epididymal sperm cells but not male fertility in an in vivo rabbit model.

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The aim of the study was to assess the effect of long exposure to summer circadian heat stress cycles on sperm parameters and the motile subpopulation structure of epididymal sperm cells from rabbit bucks. Twelve White New Zealand rabbit bucks were exposed to a daily constant temperature of the thermoneutral zone (from 18 °C to 22 °C; control group) or exposed to a summer circadian heat stress cycles (30 °C, 3 h/day; heat stress group). Spermatozoa were flushed from the epididymis and assessed for sperm quality parameters at recovery. Sperm total motility and progressivity were negatively affected by high temperatures (P < 0.05), as were also specific motility parameters (curvilinear velocity, linear velocity, mean velocity, straightness coefficient, linearity coefficient, wobble coefficient, and frequency of head displacement; P < 0.05, but not the mean amplitude of lateral head displacement). Heat stress significantly increased the percentage of less-motile sperm subpopulations, although the percentage of the high-motile subpopulation was maintained, which is consistent with the fact that no effect was detected on fertility rates. However, prolificacy was reduced in females submitted to heat stress when inseminated by control bucks. In conclusion, our results suggest that environmental high temperatures are linked to changes in the proportion of motile sperm subpopulations of the epididymis, although fertility is still preserved despite the detrimental effects of heat stress. On the other hand, prolificacy seems to be affected by the negative effects of high temperatures, especially by altering female reproduction.

Hair cortisol detection in dairy cattle by using EIA: protocol validation and correlation with faecal cortisol metabolites.

Hair may be a useful matrix to detect cumulative cortisol concentrations in studies of animal welfare and chronic stress. The aim of this study was to validate a protocol for cortisol detection in hair from dairy cattle by enzyme immunoassay (EIA). Seventeen adult Holstein-Friesian dairy cows were used during the milking period. Hair cortisol concentration was assessed in 25-day-old hair samples taken from the frontal region of the head, analysing black and white coloured hair separately. Concentrations of cortisol metabolites were determined in faeces collected twice a week during the same period of time. There was a high correlation between cortisol values in faeces and cortisol in white colour hair samples but such correlation was not significant with the black colour hair samples. The intra- and inter-assay coefficients of variation were 4.9% and 10.6%, respectively. The linearity showed R 2=0.98 and mean percentage error of -10.8 ± 1.55%. The extraction efficiency was 89.0 ± 23.52% and the parallelism test showed similar slopes. Cortisol detection in hair by using EIA seems to be a valid method to represent long-term circulating cortisol levels in dairy cattle.

Absence of beneficial effects on rabbit sperm cell cryopreservation by several antioxidant agents.

The generation of reactive oxygen species associated with cryopreservation could be responsible for mammalian sperm damage and the limitable value of stored semen in artificial insemination. The aim of this study was to assess several antioxidant agents supplemented in a commercial freezing extender (Gent B®) in order to improve post-thaw rabbit sperm quality. Ejaculates of 26 New Zealand White rabbit bucks were collected, evaluated and frozen using a conventional protocol. Antioxidant agents were tested at different concentrations: bovine serum albumin (BSA; 5, 30 or 60 mg/ml), retinol (RO; 50, 100 or 200 µM) and retinyl (RI; 0.282 or 2.82 µg/ml). Per cent viability, morphological abnormalities and intact acrosomes were determined using eosin-nigrosin staining. Motility and progressivity were analyzed by computer-assisted sperm analysis (CASA). In general, all sperm quality parameters were negatively affected by the cryopreservation process, the largest effect seen was for total motility. The addition of antioxidant agents did not improve thaw sperm quality. Furthermore, for RI groups a significant decrease in sperm quality parameters was recorded. In conclusion, rabbit sperm quality is negatively affected by the cryopreservation process. To our knowledge this report is the first using these antioxidants to supplement rabbit freezing extender. BSA and RO at concentrations used in the study did not improve sperm quality parameters after thawing, whereas RI supplementation appeared to be toxic. More studies are required to find the appropriate antioxidants necessary and their most effective concentrations to improve rabbit post-thaw sperm quality.

Feather corticosterone evaluated by ELISA in broilers: a potential tool to evaluate broiler welfare.

The measure of corticosterone (CORT) in feathers has been recently recognized as a valid and easily obtainable measure of chronic glucocorticoids secretion in avian species. This measure provides meaningful interpretations of how individuals respond to environmental perturbations. The growing interest of the public toward animal-food production welfare shows the need for improving and expanding objective tools to evaluate this issue. The present study evaluates whether it is possible to detect CORT in broiler feathers, and thus, assess if it would be a useful measure to study broiler welfare. Twenty-two broilers were randomly selected from an intensive farm. Four to 6 dorsal feathers were collected from each bird, and sex, weight, and morphological aspects of feather status were recorded. We tested the feasibility for detecting CORT in broiler feathers by ELISA, which had never been done before, and an assay validation test was performed. No significant relationships were found between feather CORT concentrations and physiological variables such as sex, weight, and fault bars in broilers. To our knowledge, this is the first study that uses broiler feathers as a matrix that provides a retrospective record of their hypothalamic-pituitary-adrenal activity. Results indicate that ELISA is a valid tool to detect feather CORT levels in broilers.

Detection of glycosylated proteins in rabbit oviductal isthmus and uterine endometrium during early embryo development.

In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of oviductal isthmus and uterine endometrium in rabbits during early embryo development. Rabbit embryos are surrounded not only by the zona pellucida but also by tubal secretion-derived mucinous coat material, the mucin coat. Twenty sexually mature females were euthanized at 0 (pre-ovulatory group) and 24, 72 and 96 h after insemination (pseudopregnancy group). The following lectin-binding agents were used: Arachis hypogaea, Peanut (PNA) to label galactosyl (ß-1,3)N- acetyl-galactosamine, Dolichos biflorus Agglutinin (DBA) to label galactosyl (ß-1,4)N- acetyl-galactosamine, Lens curinaris (LCA) to label α--mannose, α-d-glucose and Pisum sativum agglutinin (PSA) to label α-d-mannose, α-d-glucose. Blood was collected by cardiac puncture, and direct enzyme immunoassay technique was used to measure progesterone concentration. A significant increase in total plasma progesterone concentrations was detected at 96 h post-ovulation when compared with 0, 24 and 72 h post-ovulation (2.9 ± 0.5 vs 0.5 ± 0.15, 1.6 ± 0.5 and 1.5 ± 0.4 ng/ml, at 96 h vs 0, 24 and 72 h post-ovulation, respectively). No differences between pre-ovulatory and pseudopregnant females were observed for glycoprotein localization in isthmus. In contrast, in the endometrium, differences in the glycoprotein detection between pre-ovulatory and pseudopregnant stages were detected. PNA to label galactosyl (ß-1,3)N- acetyl-galactosamine was not detected at the pre-ovulatory stage, but its presence was detected at 24 h after ovulation. Both PSA and LCA to label α-d-mannose, α-d-glucose were only detected at 72 h after ovulation. DBA detection was similar for all stages of the reproductive cycle. Therefore, N-acetyl-galactosamine secreted from isthmus could be involved in the formation of the embryonic mucin coat. d-galactose (PNA), d-glucose and d-mannose (PSA and LCA) might be crucial for the implantation period.

Use of infrared thermography to assess the influence of high environmental temperature on rabbits.

The aim of this work was to ascertain if infrared thermography (IRT) can be used on rabbits to assess differences in surface body temperature when they are subjected to two different environmental temperatures outside the comfort zone. Rabbits housed in room A were maintained at a temperature of below 30°C and rabbits in room B at a temperature of above 32°C for a year. Faeces were collected six times during the year to assess stress by means of faecal cortisol metabolites (FCM). The assessment of IRT was carried out to assess maximum and minimum temperatures on the eyes, nose and ears. FCM concentration was higher in room B than A, to confirm that stress conditions were higher in room B. Significant differences in IRT were found between the animals housed in both rooms. It was observed that it was more difficult for animals from room B to maintain a regular heat loss. Although all the body zones used to assess temperature with IRT gave statistical differences, the correlations found between the eyes, nose and ears were moderate, suggesting that they were giving different information. In addition, differences up to 3.36°C were found in the eye temperature of rabbits housed in the same room, with a clear effect of their position in relation to extractors and heating equipments. Therefore, IRT could be a good tool to assess heat stress in animals housed on typical rabbit farm buildings, giving a measure of how the animal is perceiving a combination of humidity, temperature and ventilation. Some face areas were better for analysing images. Minimum temperature on eyes and temperatures on nose are suggested to assess heat losses and critical areas of the farm for heat stress in rabbits.

Retinol improves in vitro oocyte nuclear maturation under heat stress in heifers.

Heat stress (HS) is especially harmful for bovine ovarian follicle development and oocyte competence. Furthermore, HS causes premature aging in oocytes due to high levels of reactive oxygen species (ROS), involved in the harmful effects over the oocyte maturation and the steroidogenic activity of follicular cells. In this study, the presumptive protective effects of antioxidant agents on heat-stressed oocytes were evaluated. Heifer oocytes were matured for 22 h under control (38°C) and HS conditions (41.5°C at 18-21 h of maturation). For each oocyte, nuclear stage and cortical granule (CG) distribution were evaluated. Steroidogenic activity of cumulus cells was also recorded. The antioxidant agents used in the study were: retinol (1.43 µg/ml), retinyl (0.28 µg/ml) and oleic acid (0.05 mg/ml). Based on a chi-squared test (P < 0.05), HS affected negatively the metaphase II (MII) progression and produced a premature CG exocytosis. Retinol improved the oocyte MII progression. However, retinyl and oleic acid, at the concentrations used in this study, could not counteract adverse effects of HS. A decrease in progesterone and increase in estradiol availability were observed when retinyl and oleic acid were supplemented to the maturation medium, respectively. In conclusion, retinol proved to be valuable in heat-stressed oocytes protecting nuclear maturation.

Ovarian response and embryo gene expression patterns after nonsuperovulatory gonadotropin stimulation in primiparous rabbits does.

Ovarian stimulation with equine chorionic gonadotropin (eCG) is largely used in animal reproductive technologies to provide a larger number of oocytes and embryos and to improve the reproductive outcome. However, the consequences of maternal treatment with eCG on embryo gene expression patterns are not widely studied. The aim of this work was to assess the ovarian response (preovulatory follicular population, oocyte maturation, ovulation rate, and serum steroid concentrations), the early embryo survival and gene expression patterns of a panel of quality-genes involved in glucose intake, oxidative stress, apoptosis, proliferation, implantation, and fetal growth in embryos of lactating rabbits treated with eCG. A total of 34 primiparous rabbit does (Oryctolagus cuniculus) were randomly distributed at Day 23 postpartum into a treatment group receiving a unique nonsuperovulatory dose (25 IU) of eCG (eCG group; N = 17 does); or a control group without eCG treatment previously to artificial insemination (control group; N = 17 does). After 48 hours, 8 does of each group were euthanized and their ovarian response was studied. The rest of animals were artificially inseminated and their ovulation was induced with a GnRH analogue. Embryos were recovered 3.5 days later. The oocytes retrieved for in vitro maturation showed no differences in metaphase II rate in both experimental groups, although oocyte cytoplasmic maturation, in terms of cortical granule migration rate, was improved in eCG-treated does (P < 0.05). The mean number of preovulatory follicles was similar between groups but the ovulation rate was significantly higher in eCG-treated does compared with does not stimulated (P < 0.05). No differences were found in serum estradiol and progesterone concentrations of does the day of oocyte and embryo recovery, respectively. However, progesterone:estradiol ratio was slightly increased in eCG group on embryo recovery day (P = 0.1). The percentage of embryos recovered at the blastocyst stage was also increased in eCG-treated does (P < 0.05), nevertheless, there were no differences in the gene expression patterns of candidate genes SLC2A4, IGF1R, IGF2R, SHC1-SHC, TP53, PTGS2, and PLAC8; except for the transcripts of SOD1 mRNA which were downregulated in eCG-derived embryos (P < 0.05). In conclusion, the administration of eCG improves ovulation rate, oocyte cytoplasmic maturation, and blastocyst formation in primiparous rabbit does inseminated on Day 25 postpartum. Although it seems not to influence the gene expression patterns studied, a lower antioxidant defense of embryos developed after the maternal eCG treatment is suggested.

Bovine oocytes show a higher tolerance to heat shock in the warm compared with the cold season of the year.

Heat stress is especially harmful for bovine ovarian follicle development and oocyte competence. In this study, we assessed the effects of heat shock on oocyte maturation in oocytes collected during the cold (February-March; n = 114) or warm (May-June; n = 116) periods of the year. In both cases, cumulus-oocyte complexes were matured under control (38 °C) and heat shock conditions (41.5 °C, 18-21 h of maturation). For each oocyte, nuclear stage, cortical granule distribution and steroidogenic activity of cumulus cells were evaluated. Based on the odds ratio, heat-shocked oocytes were 26.83 times more likely to show an anomalous metaphase II morphology. When matured under heat shock conditions, oocytes obtained in both seasons were similarly affected in terms of nuclear maturation, whereas a seasonal effect was observed on cytoplasmic maturation. For oocytes collected during the cold season, the likelihood to show an anomalous maturation was 25.96 times higher when exposed to the heat treatment than when matured under control conditions. By contrast, oocytes collected during the warm season matured under control or heat shock did not show significant risk of showing an anomalous cytoplasmic maturation. Our findings indicate an increased rate of premature oocytes in response to heat shock as well as a higher tolerance to this stress of oocytes harvested in the warm season compared with those collected in the colder period.