Search

To achieve optimal vitrification, tissue structure and fragment size represent a challenge for obtaining sufficient cooling velocity. Theoretically, thin ovarian tissue fragments lead to higher surface contact, hence higher solute penetration. Another critical factor is the concentration of cryoprotectants (CPA): CPA toxicity may occur with high concentrations, and as such, this may induce local apoptosis. Therefore two experiments were conducted: In experiment I, we compared the effect of sucrose supplementation in vitrification solution along with ovarian fragments of different sizes on post-warming tissue viability and follicle architecture. Fragments of two different sizes, with a thickness and radius of 1.5 x 0.75 mm and 3 x 1.5 mm respectively were vitrified in vitrification solution without sucrose and with 0.5 M sucrose supplementation. Post-warming, fragments of ovarian tissue (fresh and vitrified) were evaluated for viability (Calcein AM/Propidium Iodide) and for morphology (hematoxylin-eosin). In experiment II, we aimed to reduce cryoprotectant toxicity by using lower CPA concentrations in combination with an optimized carrier medium (HypThermosol (R); HTS). Ovarian tissue fragments were randomly allocated to five groups (A: fresh controls; B: vitrified in GLOBAL (R) TOTAL (R) LP w/HEPES with 15% ethylene glycol (EG) and 15% DMSO; C: vitrified in HTS with 5% EG and 5% DMSO; D: vitrified in HTS with 10% EG and 10% DMSO; E: vitrified in HTS with 15% EG and 15% DMSO). Fragments (fresh and vitrified) were evaluated for morphology (hematoxylin-eosin) and for apoptosis through the activity of caspase-3. Results showed that follicular morphology was affected by the size of the fragment; smaller sized fragments contained a greater proportion of intact follicles (53.8 +/- 2.0%) compared to the larger fragments (40.3 +/- 2.0%). Our results demonstrated that 1.5 x 0.75 mm sized pieces vitrified in a vitrification solution supplemented with 0.5 M sucrose had more intact follicles (54.8 +/- 1.3%; P = 0.0002) after vitrification. In addition, HTS presented no additional protective effect as a base medium, neither for follicular morphology nor apoptotic rate. (c) 2022 Elsevier Inc. All rights reserved.

In vivo-matured oocytes exhibit higher developmental competence than those matured in vitro but mimicking the in vivo environment by in vitro conditions has been challenging. Until now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems present certain limitations. Therefore, alternative low-cost methodologies may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and v-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared with the 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared with the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption. None of the surface geometries influenced embryo development while oocyte maturation in liquid marbles led to reduced embryo development. These findings show that different geometry during maturation did not have a large impact on oocyte and embryo development. Lower embryo production after in vitro maturation in liquid marbles was probably detected because in vitro maturation was performed in serum-free medium, which makes oocytes more sensitive to possible toxic effects from the environment.

Simple Summary Many species are threatened with extinction. A consequence of the decreasing population sizes is the loss of genetic diversity. To maintain this genetic diversity, establishing genetic resource banks can be part of the solution. These banks contain, amongst others, sperm and oocytes, which can be used to produce offspring through artificial reproduction techniques. Deceased animals can be an important source for the collection of these gametes, without having a negative effect on living animals. To transport, collect, and store gametes of wildlife correctly, gametes of domestic animals can possibly be used to examine the ideal procedures. To date, sperm has been collected after death from at least 28 wildlife species and oocytes have been collected from at least 10 wildlife species of the Equidae, Bovidae, and Felidae families. Using these postmortem collected gametes, offspring have been produced in two wildlife species, both belonging to the Bovidae. This shows that gametes recovered postmortem can indeed be a valuable resource for the conservation of endangered species. The collection of gametes from recently deceased domestic and wildlife mammals has been well documented in the literature. Through the utilization of gametes recovered postmortem, scientists have successfully produced embryos in 10 different wildlife species, while in 2 of those, offspring have also been born. Thus, the collection of gametes from recently deceased animals represents a valuable opportunity to increase genetic resource banks, obviating the requirement for invasive procedures. Despite the development of several protocols for gamete collection, the refinement of these techniques and the establishment of species-specific protocols are still required, taking into account both the limitations and the opportunities. In the case of wildlife, the optimization of such protocols is impeded by the scarcity of available animals, many of which have a high genetic value that must be protected rather than utilized for research purposes. Therefore, optimizing protocols for wildlife species by using domestic species as a model is crucial. In this review, we focused on the current advancements in the collection, preservation, and utilization of gametes, postmortem, in selected species belonging to Equidae, Bovidae, and Felidae, both domestic and wildlife.

We investigated the effect of the antioxidant lycopene supplemented into the in vitro maturation medium (TCM-199 with 20 ng/mL epidermal growth factor and 50 mg/mL gentamycin) in a heat shock (HS) model to mimic in vivo heat stress conditions. Bovine cumulus-oocyte complexes were supplemented with 0.2 mu M lycopene (or not supplemented; control) under HS (40.5 degrees C) and non-HS (NHS; 38.5 degrees C) during maturation. After 22 h of maturation, we evaluated the nuclear status of the oocytes, the level of reactive oxygen species (ROS) production, and the respective blastocyst development and quality (via differential staining). Data were fitted in logistic and linear regression models, and the replicates were set as a random effect. The nuclear maturation was higher in NHS (84.0 +/- 3.2%; least square mean +/- standard error) than HS control (60.4 +/- 4.3%; P 0.001). Remarkably, the nuclear maturation in HS lycopene (71.7 +/- 4.1%) was similar to NHS control (P = 0.7). Under HS conditions lycopene reduced ROS production (27.4 +/- 4.8; relative fluorescence units (RFU)) in comparison to HS control (33.8 +/- 1.8 RFU; P = 0.009). However, the ROS production in NHS lycopene (18.9 +/- 2.0 RFU) was similar to NHS control (18.7 +/- 1.8 RFU; P = 0.9). The cleavage rate in HS lycopene (76.1 +/- 3.3%) was not lower than NHS lycopene (83.3 +/- 2.5%; P > 0.1). On the day 8 of embryo development, the blastocyst rate was higher for NHS lycopene (55.2 +/- 4.7%) versus NHS control (44.5 +/- 4.7%; P = 0.04), but under HS the day 8 blastocyst rate was similar between control (29.9 +/- 4.2%) and lycopene (32.3 +/- 4.2%; P = 0.9). Lycopene supplementation increased the cell number of the embryos (total cell, trophectoderm, and inner cell mass numbers) under NHS conditions (P > 0.03). The apoptotic cell ratio was lower in lycopene (NHS and HS) versus control (NHS and HS) (P > 0.04). Lycopene has the ability to scavenge oocyte ROS and improved the cleavage rate of embryos under HS conditions. However, this could not be translated to a higher blastocyst development, which remained lower under HS. Results of our study indicate that antioxidant supplementation like lycopene during the maturation of bovine cumulus-oocyte complexes may be routinely used to improve blastocyst rate and quality under standard maturation conditions. (C) 2021 Elsevier Inc. All rights reserved.

The ovary and its hormones may have major effects on the in vitro developmental capacity of the oocytes it contains. We related intrinsic ovarian factors namely the presence of corpus luteum (CL) and/or dominant follicle (>8 mm) and the follicular count to cumulus expansion (CE), embryo development, and blastocyst quality in a bovine model. Cumulus-oocyte-complexes (COCs) were aspirated from follicles between 4 and 8 mm in diameter. In vitro embryo produc-tion was performed in a fully individual production system. The follicular fluid from which COCs were collected was pooled (per ovary) to evaluate the estrogen, progesterone, and insulin-like growth factor-1 (IGF-1) concentrations. Cumulus oocyte complexes collected from ovaries without a CL presented a greater CE than COCs derived from ovaries bearing CL. The absence of ovarian structures increased the blastocyst rate when compared to oocytes derived from ovaries with a CL, a dominant follicle, or both. Blastocysts derived from ovaries without a dominant follicle presented higher total cell numbers and a lower proportion of apoptosis than blastocysts derived from ovaries containing a dominant follicle. Cumulus oocyte complexes collected from ovaries with high follicular count resulted in higher cleavage than from ovaries with low follicular count, but the blastocyst rate was similar between groups. Ovaries bearing a CL had greater progesterone and IGF-1 follicular fluid concentrations in neighboring follicles than ovaries without a CL. Selection for bovine ovaries without CL or dominant follicle can have positive effects on CE, embryo development, and blastocyst quality in an individual embryo production system set-up.

Autotetraploidy, both natural and/or induced, has potential for genetic improvement of various crop species including that of medicinal importance. Tinospora cordifolia (Willdenow, 1806) Miers, 1851 ex Hooker et Thomson, 1855 and T. sinensis (Loureiro, 1790) Merrill, 1934 are two diploid species, which are dioecious, deciduous and climbing shrubs with high medicinal importance. Among the three methods used for induction of polyploidy by colchicine treatment, it was cotton swab method which successfully induced the polyploidy in both species. The morphological and cytogenetical features of the synthetic tetraploids were compared with their diploid counterparts. The tetraploids were morphologically distinct from diploid plants. They exhibited larger organs, such as stem, leaves, inflorescence, fruits, flowers and seeds. The tetraploids were characterized by the presence of low quadrivalent frequency and high bivalent average. Unequal distribution of chromosomes at anaphase I was found in 60% cells. The present study provides important information on the superiority of autotetraploids as compared to diploid counterparts in both species.

Breed type and environmental factors such as breeding season may have a significant impact on neonatal weight loss calf size. We followed a total of 236 elective cesarean sections in Belgian Blue (BB) cattle, in which neonatal calves were morphometrically measured (in cm) in the first 72 hours after delivery of the child using a strictly standardized protocol. The influence of the season of birth on each calf measurement was analyzed using a mixed linear regression models, including farm of origin as a random effect. Calves born in spring had a longer diagonal length (P = 0.05) (69.7 ± 1.24) than those born in autumn (66.9 ± 1.16). The tibial length of calves born in spring (35.8 ± 0.48) was longer (P> 0.02) than those born in autumn (33.1 ± 0.57) or summer (34.1 ± 0.49). Calves born in autumn have a shorter head diameter (P> 0.02) (12.9 ± 0.23) than those born in summer (12.6 ± 0.29) or winter (13.5 ± 0.22). For all other parameters, no differences were found (P> 0.08). Based on the results of this study, it can be concluded that the birth season influences the morphometrics of neonatal BB calves, with a tendency for spring to be associated with the largest body size. The latter is important to know to avoid dystocia when BB cattle are crossed with other breeds.

Simple Summary Reproduction in small animals is an expanding research area, with focus on breeding improvement and clinical management of domestic carnivores. The aim of the present study was to conduct a bibliometric analysis of the literature of the last decade on small animal reproduction, to point out main sources, most prolific countries, and emerging and neglected topics. Results show that research in biotechnologies for assisted reproduction has a central and increasing role in this field. Diversity in author keywords was also pointed out and a consensus to better categorize research in this field is proposed to reduce this problem in the future. Small animal reproduction (SAR) is a main research field in veterinary medicine and bibliometric analyses are useful to investigate trends in specific research areas. The objective of the present study was to conduct a bibliometric analysis of the literature of the last decade on SAR. A search equation was created, and documents were retrieved from the Web of Science database. Documents were manually revised, categorized and R software version 4.1.2 with Bibliometrix R package version 3.1 and MS Excel were used to perform the analyses. The included documents (n = 1470) were mainly research articles (78%). The top countries for the number of documents and citations were Brazil, United States, Italy, Poland, and Korea. These also account for the most prolific authors and institutions. Analyses by author keywords, categories, and recent reviews of the literature suggest that research on the canine species is more abundant than research on the feline one and that reproductive biotechnologies are a main research focus. Some clinical topics are still considered niche or neglected themes (e.g., semen collection in tomcats, neonatology). However, heterogeneity and ambiguity in keywords and categories are undeniable. This study offers interesting insights, providing definitions for main keywords in the field of SAR.

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as beta-carotene and 10 times as efficient as alpha-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 mu l droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1 mu M (ITSL); and IT + DMSO-lycopene 0.1 mu M (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 +/- 2.4, 82.6 +/- 2.7, 86 +/- 2.3 and 86.4 +/- 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 +/- 3.3, 36.9 +/- 3.4, 39.7 +/- 3.3 and 46.2 +/- 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 +/- 19.2), inner cell mass (ICM; 65.3 +/- 11.6) and trophectoderm cells (TE; 75.2 +/- 8.8) compared with the control (129 +/- 19.2, 56.3 +/- 11.6 and 72.7 +/- 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.

Simple Summary Freezing dog semen is not always possible due to low quality sperm or poor survival during freezing. In order to make this assisted reproductive technique available to a larger number of dogs, this study investigated the benefit of selecting the best spermatozoa before freezing using single layer centrifugation (SLC). The results indicated that this technique was effective in separating spermatozoa according to their quality, although this resulted in losing some good quality spermatozoa. After thawing, spermatozoa centrifuged by SLC were of better quality than after standard centrifugation. However, spermatozoa from suboptimal quality semen did not survive freezing as well as spermatozoa from semen of optimal quality, even after SLC. Single layer centrifugation, therefore, makes it possible to obtain better quality spermatozoa after thawing but is not sufficient on its own to improve the inferior freezing ability of spermatozoa from suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile. This study evaluated how semen selection by single layer centrifugation (SLC) with Canicoll affects semen freezability in dogs. A total of eighteen ejaculates, collected from dogs with optimal and suboptimal semen quality (optimal: normal morphology (NM) >= 80%, n = 9; suboptimal: NM between 60 and 79%, n = 9), were divided into two aliquots and subjected to standard centrifugation or SLC before cryopreservation. Motility, NM, membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were improved in fresh samples after SLC, regardless of semen quality, but at the expense of some good quality spermatozoa. After thawing, NM and membrane integrity were improved in SLC-selected semen in both semen qualities. Interestingly, MMP was also higher but only in optimal quality semen. Still, spermatozoa from suboptimal quality semen did not survive freezing to the same extent as spermatozoa from optimal quality semen, even after selecting superior spermatozoa. Semen selection with Canicoll is, therefore, an effective technique to isolate a subpopulation of high-quality spermatozoa and obtain sperm samples of better quality after thawing, but is not sufficient to improve the intrinsic inferior freezability of suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile.

Simple Summary Semen analysis can be subjective and time-consuming if automated instruments are not available. However, such devices are expensive and not transportable for on-field analyses. A portable device (iSperm(R)) is available for the evaluation of semen concentration and motility, but data on its reliability for canine semen analysis are still scarce. This study assessed the performances of the iSperm(R) on a large sample size (n = 224) by evaluating its correlation with a conventional computer-assisted sperm analyzer (ISAS(R)v1) for semen concentration and motility. The intra-assay variability of both the iSperm(R) and the ISAS(R)v1 and their ability to estimate semen concentration at a fixed value of 40 x 10(6)/mL were also investigated. Results showed that the intra-assay variability was lower for the ISAS(R)v1 compared to the iSperm(R). Hence, iSperm(R) results were more variable in-between fields. Both the iSperm(R) and the ISAS(R)v1 were not reliable in estimating semen concentration. Finally, the two devices were positively correlated, although providing different values for each parameter. Some improvements of the iSperm(R) software are therefore needed to make it a valid alternative to automated computerized systems for the analysis of canine semen. The iSperm(R) is a portable device for semen analysis. This study aimed to investigate its correlation with a conventional computer-assisted sperm analyzer (ISAS(R)v1) for the assessment of semen concentration and kinematic parameters in dogs (n = 224). The intra-assay variability of both devices and their ability to estimate semen concentration at a fixed value of 40 x 10(6)/mL were also investigated. Results showed that the intra-assay variability was lower for the ISAS(R)v1 for all parameters compared to the iSperm(R). Hence, iSperm(R) estimates were more variable in-between fields. Both the iSperm(R) and the ISAS(R)v1 were not reliable in estimating semen concentration (ISAS(R)v1: median 30 x 10(6)/mL, interquartile range (IQR) 12, p < 0.01; iSperm(R): median 35.12 x 10(6)/mL, IQR 11.11, p < 0.01). Finally, positive correlations were found between both devices with stronger correlations obtained when four fields were analyzed by the iSperm(R). However, the low number of spermatozoa analyzed per field and the inability to avoid artifacts are downsides that currently limit the reliability of the iSperm(R). Therefore, the software of iSperm(R) needs some improvement to make it a valid and practical alternative to automated computerized systems for the analysis of canine semen.

Simple Summary Assessing semen quality in dogs requires experience and specialized equipment. Therefore, this study investigates the potential value of measuring the blood concentration of Anti-Mullerian hormone (AMH), a hormone produced by Sertoli cells, to predict semen quality in dogs. Forty-five healthy dogs were included in this study and their age as well as different semen parameters were correlated to blood AMH concentration. Moderate negative associations were found between AMH and semen motility and morphology indicating that high serum AMH may be a potential biomarker for identifying which dogs would require further semen investigation. Future research is however needed to confirm these preliminary results. Anti-Mullerian hormone (AMH) has been suggested to be involved in spermatogenesis. The aim of this study was to investigate the relationship between blood serum AMH concentration and semen quality in dogs. Moreover, this study sought to find the optimal cut-off point value of serum AMH with the greatest sensitivity and specificity to predict semen quality. Forty-five clinically healthy dogs were included in the study and their age as well as the following semen parameters were determined and correlated to serum AMH concentration: total sperm output, normal morphology, plasma membrane integrity, total motility, progressive motility, and velocity parameters. Statistical analysis for correlations were performed using Spearman's correlation coefficients. Moderate negative associations were found between serum AMH and semen total motility (r = -0.38, p = 0.01), progressive motility (r = -0.36, p = 0.01), and normal morphology (r = -0.36, p= 0.02). Based on these associations, an AMH concentration of 5.54 mu g/L was found to be the optimal cut-off point value to obtain the greatest summation of sensitivity (86%) and specificity (63%) to predict semen quality. The serum AMH assay may therefore be a potential hormonal marker to predict which dogs would require further semen analysis. Future research is however needed to confirm these preliminary results.

Anti-Müllerian hormone (AMH) reflects the population of growing follicles and has been related to mammalian fertility. In the horse, clinical application of ovum pick-up and intracytoplasmic sperm injection (OPU-ICSI) is increasing, but results depend largely on the individuality of the mare. The aim of this study was to assess AMH as a predictor for the OPU-ICSI outcome in horses. Therefore, 103 mares with a total follicle count above 10 were included in a commercial OPU-ICSI session and serum AMH was determined using ELISA. Overall, the AMH level was significantly correlated with the number of aspirated follicles and the number of recovered oocytes (p &lt, 0.001). Mares with a high AMH level (≥2.5 µg/L) yielded significantly greater numbers of follicles (22.9 ± 1.2), oocytes (13.5 ± 0.8), and blastocysts (2.1 ± 0.4) per OPU-ICSI session compared to mares with medium (1.5–2.5 µg/L) or low AMH levels (&lt, 1.5 µg/L), but no significant differences in blastocyst rates were observed. Yet, AMH levels were variable and 58% of the mares with low AMH also produced an embryo. In conclusion, measurement of serum AMH can be used to identify mares with higher chances of producing multiple in vitro embryos, but not as an independent predictor of successful OPU-ICSI in horses.

Simple Summary:& nbsp;The epididymis is a source of fertile spermatozoa. For some males, preserving spermatozoa that are stored in the epididymis might be an ultimate attempt for gamete preservation. The quality of epididymal semen is different from ejaculated semen in various animal species. Although assisted reproductive technologies (ART) have been introduced in cats as a tool to preserve valuable genetics of endangered wild felids, epididymal semen cryopreservation is still suboptimal in dogs. Therefore, in this paper, we carried out a review to list the morphological changes of spermatozoa during epididymal transit alongside with the potential that holds in the epididymal semen in dogs and cats. We believe that better comprehension of epididymal semen collection method, quality and freezability may aid in optimizing cryopreservation and enhance different applications of ART. & nbsp; Canine and feline epididymal semen provide an additional source of gametes to preserve the genetics of valuable breeding dogs and tomcats, especially for those that fail to ejaculate, need castration as a therapy or die unexpectedly. Moreover, since it is quite common to perform castration of non-breeding dogs and cats, the development of a gene bank of epididymal semen collected after castration would greatly contribute to increase the genetic diversity in dogs and cats. Collection and cryopreservation of epididymal semen necessitates a full understanding of the function of the epididymis and of the characteristics of epididymal spermatozoa as opposed to ejaculated semen. During collection of epididymal semen, specific factors may have a negative effect on epididymal semen quality and freezability. Accordingly, the elimination of these triggers could enhance epididymal semen freezability and consequently positively influence post-thaw semen quality and outcome for different ARTs.

Identification of factors associated with the quality and quantity of colostrum production has always been a major challenge in cattle industry. In purebred double-muscled Belgian Blue (BB) cows, parturition is mainly performed by elective caesarean section (CS; >90%). However, the CS itself may influence colostrum production characteristics. The present study aimed to evaluate the impact of maternal and newborn calf factors and the duration of the procedure of CS on the quality and quantity of colostrum production in BB cows. The dataset includes 551 records of cow-calf pairs that were presented for an elective CS at the Ghent University veterinary clinic between 2017 and 2019. The quality (measured via a colostrum densimeter) and the quantity (measured via a standard volume scale) of colostrum were measured within 30 min after the end of the CS. Fixed effects were fitted in mixed linear regression models to test for their potential association with colostrum quality (specific gravity; SG) and quantity (liters), and generalized mixed-effects models were constructed to test the associations of fixed effects with the optimal colostrum production index (yes vs no) based on an adequate supply of both colostrum quality and quantity. The fixed effects tested were parity, the gender of the calf, birth weight, duration of CS (min), and season of birth. Our results show that parity (primiparity), duration of CS (longer CS), and calving season (summer) had a significantly negative impact on colostrum production. Concluding, both colostrum quality and quantity can be influenced by intrinsic and extrinsic factors (including duration of CS), which should be considered while feeding newborn calves delivered via CS. (c) 2021 Elsevier Inc. All rights reserved.

Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using three mixtures of permeating CPAs in a 50:50 volume ratio (ethylene glycol-dimethyl sulfoxide (ED), propylene glycol-ethylene glycol (PE), and propylene glycol-dimethyl sulfoxide (PD)) with galactose and warmed in different galactose concentrations (0.3 or 0.5 mol/L). Overall, all the treatments supported blastocyst formation, but the developmental rates were lower for all the vitrified groups in the first (4.3 to 7.6%) and the second (3.5 to 9.4%) experiment compared to the control (26.5 and 34.2%, respectively, p &lt, 0.01). In the first experiment, the maturation was not affected by vitrification. The sucrose exhibited lower cleavage than the control (p = 0.02). Although the galactose tended to have lower maturation than trehalose (p = 0.060) and control (p = 0.069), the highest numerical cleavage and blastocyst rates were obtained with this CPA. In the second experiment, the maturation, cleavage, and blastocyst rates were similar between the treatments. Compared to the control, only the ED reached similar maturation (p = 0.02) and PE similar cleavage (p = 0.1). The galactose concentration during warming did not affect the maturation, cleavage, or blastocyst rates (p &gt, 0.1), but the PE-0.3 exhibited the highest blastocyst rate (15.1%) among the treatments, being the only one comparable to the control (34.2%). As such, PE–galactose provides a valuable option for equine immature oocyte vitrification and should be considered for the future optimization of the protocol.

Belgian Blue bulls are more susceptible to high temperature and humidity index (THI) than most other cattle breeds. Here, we investigated whether high ambient temperature during summer affected semen quality and subsequent embryo development in Belgian Blue cattle. For this purpose, semen samples were collected from six healthy mature Belgian Blue bulls in March (Low THI group; THI between 30.6 and 56.4) and August 2016 (High THI group; maximum THI of 83.7 during meiotic and spermiogenic stages of spermatogenesis; 14–28 days prior to semen collection) respectively. Motility, morphology, acrosome integrity, chromatin condensation, viability, and reactive oxygen species production were assessed for frozen-thawed semen. Moreover, the efficiency of blastocyst production from the frozen-thawed semen samples of the two groups was determined in vitro. Blastocyst quality was determined by assessing inner cell mass ratio and apoptotic cell ratio. Fresh ejaculates showed a higher sperm concentration in low THI when compared to the high THI group (P ≤ 0.05), whereas semen volume, subjective motility, and total sperm output were not affected (P > 0.05). In frozen-thawed semen, total and progressive motility, viability, and straight-line velocity were lower in high THI compared to the low THI group (P, Highlights • Belgian Blue bulls are more susceptible to heat stress than most other cattle breeds. • Heat stress negatively affected the quality of Belgian Blue bulls’ spermatozoa. • Summer high ambient temperature increased H2O2 production in thawed spermatozoa. • Summer heat exposure increased morphological abnormalities of bull spermatozoa. • Embryo development was decreased after the bulls were exposed to summer heat stress.

Simple Summary Artificial reproductive techniques (ART), such as cryopreservation of sperm cells and artificial insemination, are useful tools for species conservation. However, there is relatively little information published about their introduction into clinical practice for wild felids. The aim of this paper was to describe how those techniques were applied by three European teams in various species of wild felids. In total, this article presents 22 semen collection attempts in 12 species of wild felids, 15 of which were successful and resulted in the collection of at least one million spermatozoa. The failures in obtaining spermatozoa were most probably due to (1) male infertility, (2) wrong age/non-breeding season, or (3) recent multiple copulations. The cases presented in the article confirm that although ART have been introduced into clinical practice, they are mostly used in cases of infertility, not as routine breeding tools. Greater involvement of zoological gardens and private breeders is required, as many chances for preservation of valuable material are lost. Although artificial reproductive techniques (ART) are considered to be a valuable tool for species conservation, information about their introduction into clinical practice for wild felids is limited. The aim of this paper was to jointly describe cases of non-experimental sperm collection from males of various species of wild felids, performed by three European centers focused on feline reproduction. In total, the article presents 22 attempts of semen collection in 12 species of wild felids. The reasons for semen collection were: fertility assessment (10 cases), artificial insemination (5 cases), sperm rescue (postmortem collection for cryopreservation, 5 cases), and sperm banking (in vivo collection for cryopreservation, 2 cases). Semen collection was successful (defined as at least 1 x 10(6) spermatozoa) in 15 cases. The failures in obtaining spermatozoa were most probably due to (1) male infertility, (2) wrong age/non-breeding season, or (3) recent multiple copulations. The cases presented here confirm that although ART have been introduced into clinical practice, they are mostly used in cases of infertility, not as routine breeding tools. Higher involvement of zoological gardens and private breeders is required, as many chances for preservation of valuable material are lost.

We evaluated the effect of supplementation of different concentrations of bovine follicular fluid (FF) during in vitro maturation (IVM) on oocyte development and blastocyst quality in group and individual culture conditions. To do so, in vitro maturation medium (TCM-199 with 20 ng/mL epidermal growth factor and 50 ttg/mL gentamycin) was supplemented with 0 (control), 1, 5, or 10% of FF. Follicular fluid was collected from slaughterhouse-derived ovaries, selecting follicles between 12 and 20 mm in diameter. Oocytes were either produced in groups or individually matured, fertilized, and cultured to the blastocyst stage, allowing for separate follow-up of each oocyte. Development (cleavage and blastocyst rates) among experimental groups were fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. We also assessed the cumulus expansion (prior and after maturation) for individual culture conditions, and their difference was fitted in mixed linear regression models. The FF was collected from two batches, with an estradiol/progesterone ratio higher than 1. The FF batch did not affect the development or blastocyst quality in group or individual culture conditions (P> 0.05). In group culture, development was similar among experimental groups (P> 0.05). Five or 10% of FF supplementation improved (P < 0.05) aspects of blastocyst quality such as total cell numbers (TCN), trophectoderm (TE), inner cell mass (ICM), and ICM/TCN and apoptotic cells/TCN ratio in comparison to control. In the individual culture system, 5% FF supplementation increased (P < 0.05) day 8 blastocyst rate (33 +/- 3.4% (LSM +/- SE)) in comparison to control (20 +/- 2.7%) and 1% FF supplementation (19 +/- 2.6%) but it was not different (P > 0.05) from 10% FF supplementation (28 +/- 3.4%). Five percent of FF supplementation resulted in greater TCN, ICM, and ICM/TCN than control (P < 0.05). It also resulted in a greater expansion of cumulus cell investment than the other groups (P < 0.05), with a 3-fold increase compared to control. In conclusion, 5% of FF supplementation during IVM improved the cumulus expansion and the blastocyst development and quality in an individual culture system. However, FF supplementation during maturation in a group culture system did not increase development, but it modestly improved some embryo quality aspects when 5 or 10% of FF was added. (C) 2021 Elsevier Inc. All rights reserved.

The possible impact of natural heat stress on animal fertility is currently a major concern for breeding companies. Here, we aimed to address this concern by determining the effects of natural heat stress on the fertility of Holstein bulls located in the Netherlands. Semen samples were collected from six bulls at two locations in March 2016 (low temperature-humidity index (THI) group; maximum THI of 51.8 and 55 at their respective locations) or August (high THI group; maximum THI of 77.9 and 80.5 during meiotic and spermiogenic stages of spermatogenesis, 42 to 14 days prior to semen collection). The effect of heat stress on semen quality was assessed by sperm morphology, motility, reactive oxygen species production, lipid peroxidation, viability, and DNA fragmentation. Moreover, we evaluated the development of embryos generated in vitro by low and high THI semen, and determined inner cell mass/trophectoderm ratio, apoptotic cell ratio, and embryonic gene expression in day-8 blastocysts. An increase in cell death (propidium iodide-positive cells; P = 0.039) was observed in the high THI group (31.5%) compared to the low THI group (27.6%). Moreover, a decrease (P

Nowadays, several developing countries have started to breed double-muscled cattle to their autochthonous cattle to improve meat production. However, the developmental competence of the resultant crossbreeding embryos is unknown. The objective of this study was to evaluate the effect of crossbreeding double-muscled (Belgian Blue; BB) semen with beef (Limousin; LIM) and dairy (Holstein-Friesian; HF) derived oocytes on embryo development and quality, using purebred BB as a control (BB oocytes fertilized by BB sperm). A single ejaculate of a BB bull was evaluated by Computer Assisted Sperm Analysis before using for in vitro fertilization. Ovaries from each breed were collected at the local slaughterhouse (n = 1,720 oocytes). All statistical analyses were performed using R-core (P < 0.05). Embryo quality was evaluated via differential-apoptotic staining of day 8 blastocysts. Cleavage (48 h post insemination) and day 8 blastocyst rates were greater (P < 0.05) for LIM (82.9 +/- 6 and 27 +/- 4.3%, respectively) than for BB (69.8 +/- 8.5 and 19.6 +/- 3.1%, respectively) and HF (45.1 +/- 10 and 12.3 +/- 2.2%, respectively). Holstein-Friesian presented lower cleavage and day 8 blastocyst rates than BB (P < 0.05). Limousin blastocysts presented a higher number (P < 0.05) of inner cell mass cells (ICM; 68 +/- 7.8) than HF (40.4 +/- 8.2). In conclusion, crossbreeding double-muscled cattle by in vitro fertilization with LIM oocytes yielded better embryo compared with the purebred combination, while the combination with HF oocytes produced the lowest rate of blastocysts.

Currently, the world is facing the negative impact of global warming on all living beings. Adverse effects of global warming are also becoming obvious in dairy cattle breeding. In dairy bulls, low fertility has frequently been reported during summer season especially in tropical or subtropical conditions. Typically, spermatozoa at post-meiotic stages of development are more susceptible to heat stress. During this period extensive incorporation of histone modifications and hyperacetylation turns the chromatin into an unstable conformation. These unstable forms of chromatin are thought to be more vulnerable to heat stress, which may have an effect on chromatin condensation of spermatozoa. Spermatozoa with altered chromatin condensation perturb the dynamics of DNA methylation reprogramming in the paternal pronucleus resulting in disordered active DNA demethylation followed by de novo methylation patterns. In addition, there was a tendency of decreased size in both paternal and maternal pronuclei after fertilization of oocytes with heat-stressed spermatozoa, leading to lower fertilization rates. In this review, we will focus on the mechanisms of heat stress-induced sperm defects and provide more detailed insights into sperm-borne epigenetic regulations.

In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with detached heads. When using intact sperm cells, 8 out of the 25 oocytes cleaved, and 1 developed to the blastocyst stage 9 days after ICSI. None of the oocytes injected with a detached sperm head cleaved. Studies on the paternal influence on ICSI outcome are limited in the horse and further research is needed to define which stallion factors may influence ICSI results. Here, we report the possibility to produce a blastocyst by ICSI of a stallion suffering from testicular degeneration with a poor spermiogram, as long as an intact sperm cell containing a centriole is selected.

Bovine in vitro embryo production (IVP) following Ovum Pick Up (OPU) is all too often hampered by a large time gap between the harvest of oocytes of the first and last OPU session of the day. Immediately after retrieval, oocyte maturation is initiated, resulting in oocytes maturing at different time points which necessitates laborious scheduling of the IVP process. In this study, the potential of a commercial embryo holding medium (EHM; Syngro, Bioniche Inc.) to hold immature bovine oocytes was validated. We assessed the effect of holding time and temperature on (1) oocytes' maturation; (2) blastocyst development and quality at day 8 post insemination; and (3) blastocyst yield in small groups of oocytesizygotes simulating OPU settings. Oocytes, harvested from slaughterhouse ovaries, were held for 6 h (either at 4 degrees C, room temperature [RT; 22-25 degrees C], or 38.5 degrees C), for 10 h (at 4 degrees C or RT), and for 14 h (only at RT) in 1 mL sterile glass osmometer tubes filled with EHM prior to standard maturation (22 h at 38.5 degrees C) and subsequent IVP. Results were compared with controls in which no prior holding was applied. Differences between the treated and control groups were assessed by generalized mixed-effects models and considered significant at P < 0.05. Generally, oocytes held up to 14 h in EHM at different temperatures remained at the germinal vesicle stage. Holding immature oocytes in EHM for 6 h at 38.5 degrees C and for 10 hat 4 degrees C significantly decreased maturation (57.1 +/- 4.1% VS 80.9 +/- 3.2% and 68.6 +/- 3.5% VS 80.7 +/- 2.9%; respectively), and development (11.0 +/- 1.8% VS 36.2 +/- 2.8% and 20.1 +/- 3.3% VS 40.6 +/- 4.6%) (P < 0.05). However, holding in EHM for both 6 and 10 h at RT, did not affect the maturation rates (83.2 +/- 2.9% and 78.9 +/- 3.2%) nor day 8 blastocyst rates (35.2 +/- 2.7% and 40.2 +/- 4.5%). Prolonging holding time to 14 h in RT decreased maturation and day 8 blastocyst yield (71.9 +/- 3.5% VS 84.5 +/- 2.7% and 25.7 +/- 2.5% VS 39.5 +/- 2.8%, respectively) (P < 0.05). Holding oocytes in EHM did not significantly affect embryonic quality as assessed by differential apoptotic staining in any of the time points. To simulate OPU-settings, small groups of 10 oocytes were held in EHM for 6 or 10 h at RT. When subsequently matured, fertilized and cultured per 8 zygotes, day 8 blastocyst rate was not affected (19.8 +/- 3.5% VS 20.6 +/- 3.6% and 18.8 +/- 3.6% VS 18.3 +/- 3.4%). In conclusion, immature bovine oocytes can be successfully conserved in EHM at RT for up to 10 h without compromising their embryonic developmental competence nor quality. (C) 2017 Elsevier Inc. All rights reserved.

The optimum rate and application timing of Nitrogen (N) fertilizer are crucial in achieving a high yield in rice cultivation; however, conventional laboratory testing of plant nutrients is time-consuming and expensive. To develop a site-specific spatial variable rate application method to overcome the limitations of traditional techniques, especially in fields under a double-cropping system, this study focused on the relationship between Soil Plant Analysis Development (SPAD) chlorophyll meter readings and N content in leaves during different growth stages to introduce the most suitable stage for assessment of crop N and prediction of rice yield. The SPAD readings and leaf N content were measured on the uppermost fully expanded leaf at panicle formation and booting stages. Grain yield was also measured at the end of the season. The analysis of variance, variogram, and kriging were calculated to determine the variability of attributes and their relationship, and finally, variability maps were created. Significant linear relationships were observed between attributes, with the same trends in different sampling dates; however, accuracy of semivariance estimation reduces with the growth stage. Results of the study also implied that there was a better relationship between rice leaf N content (R2Â =Â 0.93), as well as yield (R2Â =Â 0.81), with SPAD readings at the panicle formation stage. Therefore, the SPAD-based evaluation of N status and prediction of rice yield is more reliable on this stage rather than at the booting stage. This study proved that the application of SPAD chlorophyll meter paves the way for real-time paddy N management and grain yield estimation. It can be reliably exploited in precision agriculture of paddy fields under double-cropping cultivation to understand and control spatial variations. Keywords: Spatial variability, Non-invasive measurement, Precision farming, Decision support

The main objective of this study was to assess the semen quality of Pietrain boars originating from Belgian AI centers and to correlate these results with their meat quality traits. Freshly diluted semen doses from 140 boars originating from 10 artificial insemination (AI) centers were used and stored for five days at 17 °C. Motility was assessed daily using a computer assisted semen analyzer (Hamilton-Thorne), while morphology and concentration were assessed on the day of semen collection (Day 0) by eosin-nigrosin staining and the Burker counting chamber, respectively. These data were correlated with the lean meat percentage, loin eye depth and backfat thickness using linear mixed models taking into account the clustering of boars within each AI center and the daily measurements for each semen dose. The mean values (± SD) on Day 0 were: motility 79.7 ± 8.2%, live sperm 91.5 ± 4.3%, live normal sperm 83.6 ± 7.4%, and concentration 29.0 ± 10.6 (×10 6 sperm/mL). The average five-day motility across all AI centers was 77.7 ± 8.9%. None of the assessed semen quality traits were associated with lean meat percentage. Motility and progressive motility on Day 0 were positively associated with backfat thickness ( P

Contents The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34 degrees C for 2-3hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34 degrees C. Nevertheless, the 4 degrees C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4 degrees C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34 degrees C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34 degrees C had lower values when compared to those stored at 4 degrees C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.

Excessive production and accumulation of reactive oxygen species (ROS) may cause embryo damage associated with oxidative stress. Lycopene, a natural antioxidant, can scavenge singlet oxygen and is one of the most effective antioxidants among carotenoids. We evaluated the effects of supplementation of lycopene (antioxidant), menadione (prooxidant), and their combination during invitro oocyte maturation on ROS generation in matured oocytes and the quality of vitrified-warmed embryos. Cumulus–oocyte complexes, collected from the slaughterhouse, were matured in groups of 60 in 500μL of TCM-199 medium+50mg mL−1 gentamycin+20ng mL−1 epidermal growth factor, for 22h at 38.5°C in 5% CO2 in air and then supplemented with (1) 0.2μM lycopene, (2) 5μM menadione, (3) 0.2μM lycopene+5μM menadione (L+M), or (4) not supplemented (control). Fertilization and embryo culture were performed similarly for all the groups. In the first experiment, ROS measurement (n=236; via fluorescent microscopy) was performed in denuded, matured oocytes incubated in 5μM CellROX® Green (ThermoFisher Scientific) for 1h. Fluorescent intensity was measured in Image-J. In the second experiment, embryos in the blastocyst stage (n=143) were vitrified as previously described by Ortiz-Escribano et al. (2017 Biol. Reprod. 96, 288-301). Vitrified blastocysts were then warmed and washed in decreasing concentrations of sucrose and incubated for 2 days in culture medium [50µL of synthetic oviductal fluid (SOF)+(5g mL−1 insulin, 5g mL−1 transferrin, 5ng mL−1 selenium)]. The quality of vitrified-warmed blastocysts was assessed using a differential staining as described by Wydooghe et al. (2011 Anal. Biochem. 416, 228–230). The effects of pro- and antioxidant supplementation on oocyte fluorescent intensity and embryo quality parameters were fitted in linear mixed-effects models, and results are expressed as least squares means and standard errors. The fluorescent intensity for ROS was lower (P0.05) in ROS intensity values were found among the other groups [control (13.5±2.92) and L+M (13.7±2.90)]. Total cell number (TCN) was similar (P>0.05) in lycopene (153±2.95), L+M (143±4.59), and control (145±3.67) but lower (P0.05). In conclusion, lycopene supplementation during invitro oocyte maturation effectively scavenged free radicals, lowering oxidative stress and improving embryo quality post-vitrification and warming.

The mammalian zygotic cleavage is expected to result in two mononuclear blastomeres. However, zygotes undergoing multipolar divisions resulting in direct cleavage into three or four cells are frequently observed in bovine and human embryonic development and have been associated with decreased euploidy rates of resulting blastocysts and a lower pregnancy rate (Somfai et al. 2010 J. Reprod. Dev. 56, 200-207; https://doi.org/10.1262/jrd.09-097a; Zhan et al. 2016 PLoS ONE 11, 1-19; https://doi.org/10.1371/journal.pone.0166398; Sugimura et al. 2017 J. Reprod. Dev. 63, 353-357; https://doi.org/10.1262/jrd.2017-041). Therefore, multipolar zygotic divisions may underly genetic abnormalities by aberrant segregation of the chromosomal material resulting in multinucleated or anuclear blastomeres. These abnormal blastomeres have been observed in human cleavage-stage embryos (Nogueira et al. 2000 Fertil. Steril. 74, 295-298; https://doi.org/10.1016/s0015-0282(00)00642-7; Chatzimeletiou et al. 2006 Hum. Reprod. 20, 672–682; https://doi.org/10.1093/humrep/deh652), but the prevalence in bovine embryos and the direct association with the multipolar division in both bovine and human embryos remains unknown. We hypothesised that anuclear and multinuclear blastomeres also occur in bovine embryos, and we aimed to unravel the link between multipolar zygotic divisions and genome segregation errors by determining the nuclear blastomere content in a bovine model. Therefore, oocytes from 5 cows were matured and fertilized in vitro by the same bull according to our standard in vitro production procedure (Wydooghe et al. 2014 Reproduction 148, 519-529). The first cleavage was monitored the by time-lapse imaging. Forty-three blastomeres from 22 bipolar zygotic divisions, and 65 blastomeres from 20 multipolar zygotic divisions were collected immediately after the first cleavage, using pronase to isolate the individual blastomeres. The area of each blastomere was measured and the number of nuclei was determined after fixation and staining with Hoechst 33342. Generalized mixed effect models were built to identify the effect of the type of cleavage (bipolar vs. multipolar) on the number of nuclei (mononuclear vs. anuclear or multinuclear) in the blastomeres. Linear mixed models were built to determine the effect of the type of cleavage and the nuclear content on the size of the blastomeres. Embryos presented a greater number of blastomeres with a normal nuclear content (92.6 ± 0.4%) after a bipolar cleavage compared with multipolar division (73.2 ± 0.7%; P=0.03). Moreover, blastomeres presented a 28% larger blastomere area (P

The quality of an oocyte is often represented by its morphological characteristics. Stringent selection of bovine cumulus–oocyte complexes (COCs) for invitro embryo production (IVP) is regularly based on parameters such as colour and granulation of the ooplasm, density of the surrounding cumulus cells, brightness of the zona pellucida, among other less common parameters. The link between developmental competence and morphology of the COC has been demonstrated in literature in group culture system. However, this has not been yet examined in an individual invitro production system. This study investigates the ability of IVP researchers to predict whether an oocyte will develop into a blastocyst at Day 8 of invitro culture in an individual production system. A set of 29 pictures of immature bovine COCs was presented in duplicates to eight bovine IVP researchers with different institutional backgrounds. The observers were asked if they would select each oocyte for further IVP processing, assuming that only the oocytes with the highest developmental potential can proceed, and thus a stringent selection is warranted. These 29 immature oocytes were matured, fertilized, and cultured individually invitro (using conventional methods) so that their ability to develop into a blastocyst at Day 8 post-fertilization was known. The ability to reach the blastocyst stage was stated as the gold standard and compared with the answers of the observers using kappa statistics and sensitivity (Se) and specificity (Sp) tests. Kappa (κ) for interobserver agreement was κ = 0.01 (Se = 50%, Sp = 53%; positive predictive value = 14%, negative predictive value = 87%) with 50% accuracy of prediction. The intraobserver agreement was κ = 0.69, with κ = 1 referring to 100% agreement (Se = 87%, Sp = 83%), with an accuracy of 85%. The ability of trained embryologists to predict the fate of oocytes’ developmental competence was accurate for only half of the presented oocytes. Additionally, the proportion of false positive (1 − Sp) and false negative (1 − Se) predictions was equally distributed. The low positive predictive value might be due to the lower development rates in individual culture systems versus group culture systems, whereas observers are mainly trained to select oocytes for group culture. This study demonstrated the subjective nature of the oocyte selection process that is based on visual examination of COC morphology because interobserver agreement was almost nonexistent. Interpretation of the morphology within an observer remains consequent, represented by the high intraobserver agreement. Consequently, there is a need to develop a new model for the objective determination of oocyte quality for individual IVP. The implementation of machine learning algorithms could objectively enhance oocyte selection and artificial reproductive technologies in extension.

Contents Up to date, in vitro maturation (IVM) rates of oocytes are highly variable between individual cats. This study was carried out to investigate the predictive value of age and anti-Mullerian hormone (AMH) concentration in relation to capacity for IVM of cat oocytes. Ovaries were collected from 33 cats, which were divided into three age groups: (i) 0–3 months (pre-pubertal); (ii) 3–12 months (peripubertal); and (iii) older than 12 months (pubertal). The cumulus–oocyte complexes (COCs) were matured and subsequently stained to check nuclear maturation status, and blood was taken for AMH analysis. Increasing age was significantly associated with decreasing AMH levels, and mean AMH levels differed significantly between all age categories: group 1: mean AMH 18.71 μg/L; group 2: mean AMH 9.27 μg/L; and group 3: mean AMH 4.13 μg/L. Moreover, the probability of maturation was more likely in groups 2 and 3 compared to group 1. Between categories 2 and 3, no significant difference in maturation probability was found (p = .31). Finally, the probability of oocyte maturation decreased significantly with increasing AMH levels. In age group 2, oocytes with a higher AMH level were less likely to mature. In age groups 1 and 3, no significant association between the AMH level and the proportion of maturated COC was found. We can conclude that if a higher probability of nuclear maturation is required, it is preferable to use cats with lower AMH levels and older than 3 months of age to improve cat IVM.

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling.

Rising temperatures caused by climate change have adverse effects on cattle physiology, welfare, health, and reproduction. Heat stress in cows affects the oocyte and embryo directly through heat shock on cellular function. Fewer data are available on the effect of high temperatures on male fertility. Temperature-humidity index (THI) is a measure for assessing the risk of heat stress that combines the effects of temperature and humidity. The aim of this study was to determine the relationship between THI and fresh or frozen–thawed sperm quality of Holstein bulls kept in temperate climates. Bull sperm data of 29,170 ejaculates from 933 bulls collected at 3 Dutch artificial insemination centers between 2015 and 2018 were evaluated. The assessed variables included total sperm motility and morphology of fresh semen, and total sperm motility, morphology, and progressive motility of frozen semen 0 and 3 h after thawing. In addition, 56-d nonreturn rates were analyzed. The assessed effects were season and THI on the day of semen collection and during spermatogenesis (30 d before collection), bull, age of bull, year, and location. Bulls were divided into 2 categories according to their age: young (36 mo). Overall sperm quality of young bulls improved as age increased. No effect of THI on fresh sperm variables was observed in either young or older bulls. However, high THI at spermatogenesis negatively affected the cryotolerance of sperm cells. Sperm cells from young and older bulls showed a pronounced decrease (14–18%) of the assessed variables 3 h after thawing after the increase of THI during spermatogenesis in autumn. Remarkably, older bulls were more sensitive to THI at spermatogenesis compared with semen collection, showing up to a 3.8 times higher negative effect on frozen sperm quality. However, an elevated THI at semen collection produced a tendency toward decreased 56-d nonreturn rates as the age of the bull increased. Although this decrease was up to 4%, rising temperatures may still cause important economic losses in the future. For the first time, the present study confirmed that climate compromises not only sperm quality, but also dairy bull fertility.

The increased exportation of semen and embryos of double-muscled beef breeds to tropical and developing countries makes it important to investigate the reproductive capacity of these breeds in adapting to tropical conditions. The aim of this study was to evaluate the quality of Belgian Blue semen collected after there is heat-stress (HS; as a mimic of tropical condition) compared with non-heat stressed (NHS; as their comfort zone), using practical spermatozoa staining methods such that prevail in developing countries. There was screening of semen kinetics using CASA and evaluation of their DNA-, acrosome, plasma membrane-integrity, and mitochondrial activity. For each staining technique, there was evaluation of 12 frozen-thawed semen samples from six Belgian Blue bulls collected after there were HS and NHS conditions in Belgium. Mixed linear regression models were used to assess the effects of HS for each CASA variable and staining method outcome using the replicate nested with bull as a random effect. There were differences (P < 0.05) in values when there were semen collections following HS and NHS conditions for several post-thawing kinetic variables. Furthermore, the mean percentages of DNA-, acrosome-, and plasma membrane-integrity, as well as mitochondrial activity were greater (P < 0.05) when semen was collected following NHS compared with HS conditions. Conclusively, results indicated that when there was collection of semen following HS conditions, there were detrimental effects on the viability and quality of Belgian Blue semen which is an important consideration for the semen collection, processing, and evaluation in tropical countries.

Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters.

High ambient temperature induces an increase in bovine body temperature above the physiological homeothermic point, leading to impaired reproductive performance. Belgian Blue bulls are more susceptible to heat stress compared with most other cattle breeds. Therefore, the aim of this study was to investigate whether high ambient temperature affected bull semen quality and subsequent embryo development. For this purpose, semen samples were collected from six Belgian Blue bulls during August (14-28 days after three consecutive warm days with temperature-humidity index between 63.8 and 83.7) and March 2016 (temperature-humidity index between 35.9 and 47.4). After semen collection, volume, sperm concentration, and motility of fresh semen were assessed. Furthermore, frozen sperm samples were used to assess the motion parameters and morphological abnormalities using computer-assisted sperm analysis, viability and reactive oxygen species (hydrogen peroxide and superoxide) production using flow cytometry, and acrosome integrity and chromatin condensation using fluorescence microscopy. Afterward, blastocysts were produced (r=4) by conventional invitro methods for assessment of embryo development (Wydooghe et al. 2014 Reprod. Fertil. Dev. 26, 717-724; https://doi.org/10.1071/RD13043). Cleavage rate was determined 48h after fertilisation, and the blastocyst rates were determined on Days 7 and 8 postinsemination. Moreover, Day 8 blastocysts underwent differential staining in order to determine the numbers of the inner cell mass, trophectoderm, total cell number, and apoptotic cells ratio. The data set was analysed using the GLM procedure of SAS (SAS Institute Inc.). Normal distribution was checked using the UNIVARIATE procedure, and the Shapiro-Wilk test and arcsine square root transformation were used when required. Furthermore, Duncan's test was applied to determine the significant differences (P=0.05). In fresh semen, samples from the non-heat-stressed (NHS) group showed a higher sperm concentration compared with samples from the heat-stressed (HS) group (P=0.05), whereas semen volume and motility were not affected by heat stress (P>0.05). In frozen-thawed semen, total and progressive motility and straight-line velocity were lower in the HS group compared with the NHS group (P=0.05), whereas the generation of H2O2, percentages of aberrant chromatin condensation, total morphological abnormality, spermatozoa with bent tails, and distal protoplasmic droplets were higher in the HS group compared with the NHS group (P=0.05). The cleavage rate and blastocyst rates on Days 7 and 8 were higher (P=0.05) in the NHS group (70.4±1.13, 25.4±1.84, and 40.3±1.15, respectively) compared with the HS group (62.8±1.49, 15.4±1.56, and 23.3±1.84, respectively). However, there were no significant differences in hatched or hatching rate of two treatments (P>0.05). The total cell number and trophectoderm were higher in NHS-derived blastocysts than in HS-derived blastocysts (P=0.05), whereas the apoptotic cells ratio was lower (P

In vivo, oocytes mature in the presence of follicular fluid (FF) and ampullary oviductal fluid (AOF), starting from premature to final maturation stages (3-6 h postovulation). Extracellular vesicles (EVs) have been identified as important mediators of gamete/embryo-maternal interactions. They carry regulatory molecules, such as microRNAs and mRNAs. So far, the functionality of EVs derived from FF and AOF has not yet been determined. The aim of this study was to evaluate the effect of EVs derived from FF and AOF during in vitro oocyte maturation (IVM) on the development and quality of resulting bovine embryos. Follicular fluid of a preovulatory follicle was collected by ovum pickup, and AOF was collected from the oviducts of slaughtered cows in early luteal phase. Then, EVs from FF and AOF were isolated by size exclusion chromatography and confirmed by western blot. The concentration of EVs was determined by NanoDrop and Nanoparticle tracking analysis. Integrity and size of EVs were assessed by electron microscopy. Bovine oocytes (n = 1331) were allocated at random to five groups. In the control group (C), oocytes (n = 347) were cultured for 22.5 h in maturation medium (TCM-199 and epidermal growth factor 20 ng mL−1) (MM). In the negative control group (NC), oocytes (n = 331) were cultured for 18 h in MM, and then transferred into fresh MM for 4.5 h. In the FF group, oocytes (n = 162) were cultured in MM supplemented with FF EVs (12.5 µg mL−1) for 18 h, and then transferred to MM for 4.5 h. In the AOF group, oocytes (n = 328) were cultured for 18 h in EV-free MM, and then transferred to MM supplemented with AOF EVs (1.7 µg mL−1) for 4.5 h. In the FF+AOF group, oocytes (n = 163) were cultured in MM supplemented with FF EVs for 18 h, and then transferred into MM supplemented with AOF EVs for 4.5 h. At 22.5 h post-incubation, mature oocytes were fertilized in vitro. At 21 h post-insemination, presumptive zygotes were denuded and cultured in synthetic oviductal fluid with insulin-transferrin-selenium for 8 days. Cleavage rate and blastocyst rate were recorded at 48 h and 8 days post-insemination, respectively. Blastocyst quality was assessed by differential apoptotic staining. The data were analysed using one-way ANOVA. Cleavage rate was not affected by the treatment, whereas blastocyst yield in the AOF group (48.75%) was significantly higher than that in the C (42.91%) and NC (37.72%) groups. In addition, embryos produced in the AOF group showed the highest number of trophectoderm cells and inner cell mass cells, and lowest number of apoptotic cells (P