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Contents Lead (Pb2+) is a toxic heavy metal which interferes with several physiological processes regulated by Ca2+, including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams ( Ovis aries) have been incubated in vitro with or without 50 ng Pb2+/ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb2+ on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb2+ have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb2+ action. On the contrary, Pb2+-incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb2+ + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb2+ in spermatozoa; thus, the enhancement of Pb2+ effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb2+ by spermatozoa. However, the increase of intracellular Pb2+ in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected. [ABSTRACT FROM AUTHOR]

In this study, we evaluated sheep sperm quality after using Tris-citrate-fructose-based extender with and without egg yolk, a Tris-citrate without fructose and with egg yolk and the commercial extender Biladyl®, preserving diluted semen at 15 and 23°C for different times (4, 24, 48 and 72 h). The results showed that the diluents with fructose and egg yolk gave the best results of seminal quality. Moreover, the production of ROS was higher for the temperature of 23°C compared to the temperature of 15°C (control). In addition, VCL and the percentage of spermatozoa with intact acrosome decreased with temperatures of 23°C. Finally, a drastic decrease in sperm quality was observed after 24 hours of preservation for most of the parameters evaluated., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Male infertility (MI) involves various endogenous and exogenous facts. These include oxidative stress (OS), which is known to alter several physiological pathways and it is estimated to be present at high levels in up to 80% of infertile men. That is why since the late 20th century, the relationship between OS and MI has been widely studied. New terms have emerged, such as Male Oxidative Stress Infertility (MOSI), which is proposed as a new category to define infertile men with high OS levels. Another important term is MOXI: Male, Antioxidants, and Infertility. This term refers to the hypothesis that antioxidants could improve male fertility without the use of assisted reproductive technology. However, there are no evidence-based antioxidant treatments that directly improve seminal parameters or birth ratio. In this regard, there is controversy about their use. While certain scientists argue against their use due to the lack of results, others support this use because of their safety profile and low price. Some uncertainties related to the use of antioxidants for treating MI are their questionable efficacy or the difficulties in knowing their correct dosage. In addition, the lack of quality methods for OS detection can lead to excessive antioxidant supplementation, resulting in "reductive stress". Another important problem is that, although the inflammatory process is interdependent and closely linked to OS, it is usually ignored. To solve these uncertainties, new trends have recently emerged. These include the use of molecules with anti-inflammatory and antioxidant potential, which are also able to specifically target the reproductive tissue; as well as the use of new methods that allow for reliable quantification of OS and a quality diagnosis. This review aims to elucidate the main uncertainties about MOXI and to outline the latest trends in research to develop effective therapies with clinically relevant outcomes.

Background: Artificial insemination (AI) is a routine breeding technology in animal reproduction. Nevertheless, the temperature-sensitive nature and short fertile lifespan of ram sperm samples hamper its use in AI. In this sense, nanotechnology is an interesting tool to improve sperm protection due to the development of nanomaterials for AI, which could be used as delivery vehicles. In this work, we explored the feasibility of vitamin E nanoemulsion (NE) for improving sperm quality during transport., Results: With the aim of evaluating this proposal, ejaculates of 7 mature rams of Manchega breed were collected by artificial vagina and extended to 60 × 10 6 spz/mL in Andromed®. Samples containing control and NE (12 mmol/L) with and without exogenous oxidative stress (100 µmol/L Fe 2+ /ascorbate) were stored at 22 and 15 ºC and motility (CASA), viability (YO-PRO/PI), acrosomal integrity (PNA-FITC/PI), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C 11  BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) monitored during 96 h. Our results show that NE could be used to maintain ram spermatozoa during transport at 15 and 22 ºC for up to 96 h, with no appreciable loss of kinematic and physiological characteristics of freshly collected samples., Conclusions: The storage of ram spermatozoa in liquid form for 2-5 d with vitamin E nanoemulsions may lead more flexibility to breeders in AI programs. In view of the potential and high versatility of these nanodevices, further studies are being carried out to assess the proposed sperm preservation medium on fertility after artificial insemination., (© 2023. The Author(s).)

Conventional DNA analysis techniques can hardly detect DNA damage in ruminant spermatozoa due to high DNA compaction in these cells. Furthermore, these techniques cannot discriminate whether the damage is due to oxidative stress. The main purpose of this study was to evaluate the efficacy of two techniques for determining DNA damage in ovine sperm when the source of that damage is oxidative stress. Semen samples from twenty Manchega rams (Ovis aries) were collected and cryopreserved. After thawing, the samples were subjected to different levels of oxidative stress, and DNA oxidation was quantified using an 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunodetection assay and Sperm Chromatin Structure Assay (SCSA®). For this purpose, we evaluated five different concentrations of an oxidation solution (H2O2/FeSO4•7H2O) on ram sperm DNA. Our study with the 8-OHdG immunodetection assay shows that there are higher values for DNA oxidation in samples that were subjected to the highest oxidative stress (8 M H2O2/800 µM FeSO4•7H2O) and those that were not exposed to high oxidative stress, but these differences were not significant (p ≥ 0.05). The two SCSA® parameters considered, DNA fragmentation index (DFI %) and high DNA stainability (HDS %), showed significant differences between samples that were subjected to high concentrations of the oxidation agent and those that were not (p < 0.05). We can conclude that the 8-OHdG immunodetection assay and SCSA® detect DNA damage caused by oxidative stress in ovine sperm under high oxidative conditions; SCSA® is a more straightforward method with more accurate results. For these reasons, an oxidative-stress-specific assay such as 8-OHdG immunodetection is not needed to measure DNA damage caused by oxidative stress in ram sperm samples.

The advent of nanotechnology in the field of animal reproduction has led to the development of safer and more efficient therapies. The use of nanotechnology allows us to avoid the detrimental effects of certain traditional antioxidants, such as Vitamin E. Its hydrophobic nature makes mandatory the use of organic solvents, which are toxic to sperm cells. This study aims to evaluate the efficiency of vitamin E nanoemulsions (NE) on ram ( Ovis aries ) spermatozoa. For this purpose, the effect of three NE concentrations (6, 12, and 24 mM) were assessed on sperm of 10 mature rams of the Manchega breed. Sperm samples were collected by artificial vagina, pooled, and diluted in Bovine Gamete Medium. The samples were stored at 37 °C and assessed at 0, 4, 8, and 24 h under oxidative stress conditions (100 µM Fe 2+ /ascorbate). Motility (CASA), viability (YO-PRO/IP), acrosomal integrity (PNA-FITC/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C 11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA ®® ) were assessed. A linear mixed-effects models were used to analyze the effects of time, NE, and oxidant (fixed factors) on sperm parameters, and a random effect on the male was also included in the model with Tukey's post hoc test. Protection of ram spermatozoa with NE resulted in a more vigorous motility under oxidative stress conditions with respect Control and Free vitamin E, while preventing the deleterious effects of oxidative stress coming from the production of free radicals and lipid peroxidation. These results ascertain the high relevance of the use of delivery systems for sperm physiology preservation in the context of assisted reproduction techniques.

Incubation with estrous sheep serum (ESS) is required to induce in vitro capacitation of spermatozoa during in vitro fertilization of small ruminants. However, the effect of adding different serum concentrations in the fertilization media on the quality of resulting blastocysts has not yet been studied. Here, 298 sheep oocytes were co-incubated with capacitated spermatozoa with either 10% or 2% ESS. There were no differences between treatments in cleavage (10% ESS: 63.81 ± 5.87% and 2% ESS: 45.31 ± 5.87%) and blastocyst rates (10% ESS: 20.83 ± 2.12% and 2% ESS: 15.93 ± 2.12%). Nonetheless, in vitro-produced blastocysts from the 10% ESS treatment showed a higher transcript abundance of mRNAs involved in apoptosis (ITM2B and BCL2), antioxidant defence (GPX1) and growth-related imprinting (IGF2R). Our data suggest that ESS supplementation during in vitro fertilization can influence the quality of sheep embryos at later stages of development by increasing the transcription of developmentally important genes., (© 2022 The Authors. Reproduction in Domestic Animals published by Wiley-VCH GmbH.)

The preservation of ovaries beyond 7 h dramatically decreases the developmental potential of oocytes to reach the blastocyst stage during in vitro embryo production. Here we investigated the protective effects of melatonin in the ovarian preservation solution after prolonged storage (7 h) in ovine as an animal model. Slaughterhouse adult sheep ovaries were preserved in saline solution for 2 h (Control) and 7 h (Control stress), and with melatonin for 7 h and at different concentrations (Melatonin 10 -3 , 10 -5 , 10 -7 , 10 -9 , and 10 -11  M). First, the fertilizing ability, embryo development rates, and blastocyst quality were investigated. Notably, a concentration of 10 -9  M melatonin showed the greatest number (p < 0.05) of blastocysts produced after 7 h of ovary storage (24.75 ± 1.57%) and was comparable (p > 0.05) to that obtained after just 2 h of storage in the untreated Control (30.77 ± 1.57%). Then, oocyte quality parameters showed that, compared to Control stress, Melatonin actively reduced intracellular ROS content, caspase-3 activity, DNA fragmentation, and the abundance of pro-apoptotic transcripts BAX and CASP3, while increasing that of GDF9 and GPX1. In cumulus cells, flow cytometry results showed that melatonin decreased apoptosis and increased mitochondrial activity (p < 0.05). In addition, there was a greater (p < 0.05) abundance of HAS2, STAR, and PTGS mRNA transcripts in Melatonin compared to Control stress. These findings reveal a melatonin-mediated developmental rescue of oocytes against ischemic damage during ovary preservation which represents a promising strategy for successfully producing embryos when prolonged ovarian transport times are required., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2022. Published by Elsevier Inc.)

Creating germplasm banks of wild species, such as the Iberian red Deer ( Cervus elaphus hispanicus ) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values ( p ≤ 0.05) for mitochondrial activity and lower values ( p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences ( p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refrigerate the sperm samples by storing them in straws with a 120 min equilibration period and freezing them in liquid nitrogen vapors in a tank or box.

The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm regions.

Oxidative stress has become a major concern in the field of spermatology, and one of the possible solutions to this acute problem would be the use of antioxidant protection; however, more studies are required in this field, as highly contradictory results regarding the addition of antioxidants have been obtained. Vitamin E is a powerful biological antioxidant, but its low stability and high hydrophobicity limit its application in spermatology, making the use of organic solvents necessary, which renders spermatozoa practically motionless. Keeping this in mind, we propose the use of hydrogels (HVEs) and nanoemulsions (NVEs), alone or in combination, as carriers for the controlled release of vitamin E, thus, improving its solubility and stability and preventing oxidative stress in sperm cells. Cryopreserved sperm from six stags was thawed and extended to 30 × 10 6 sperm/mL in Bovine Gamete Medium (BGM). Once aliquoted, the samples were incubated as follows: control, free vitamin E (1 mM), NVEs (9 mM), HVEs (1 mM), and the combination of HVEs and NVEs (H + N), with or without induced oxidative stress (100 µM Fe 2+ /ascorbate). The different treatments were analyzed after 0, 2, 5, and 24 h of incubation at 37 °C. Motility (CASA ® ), viability (YO-PRO-1/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipid peroxidation (C 11 BODIPY 581/591), intracellular reactive oxygen species production (CM-H 2 DCFDA), and DNA status (SCSA ® ) were assessed. Our results show that the deleterious effects of exogenous oxidative stress were prevented by the vitamin E-loaded carriers proposed, while the kinematic sperm parameters ( p ˂ 0.05) and sperm viability were always preserved. Moreover, the vitamin E formulations maintained and preserved mitochondrial activity, prevented sperm lipid peroxidation, and decreased reactive oxygen species (ROS) production ( p ˂ 0.05) under oxidative stress conditions. Vitamin E formulations were significantly different as regards the free vitamin E samples ( p < 0.001), whose sperm kinematic parameters drastically decreased. This is the first time that vitamin E has been formulated as hydrogels. This new formulation could be highly relevant for sperm physiology preservation, signifying an excellent approach against sperm oxidative damage.

Oocyte quality is crucial for subsequent embryo development and so it is a major challenge in assisted reproductive technologies. The aim of the present work was to evaluate the morphometric parameters of oocytes (experiment 1) and the relative gene expression of oocytes and cumulus cells (CCs) (experiment 2) as biomarkers of oocyte quality after individually culturing them (one oocyte or embryo/drop). In experiment 1, individually matured oocytes were measured and classified into small, intermediate, and large oocytes after a cluster analysis, based on total diameter (with zona pellucida, ZP), oocyte diameter (without ZP), and ZP thickness. These oocytes were individually fertilized in vitro and cultured. The embryo development was evaluated up to the blastocyst stage. According to the total diameter, oocyte diameter, and ZP thickness, the blastocyst rate decreased in the small oocytes group (3.1 ± 3.1, 14.1 ± 9.4, and 26.7 ± 3.9, respectively) compared to the intermediate (29.4 ± 5.2, 30.5 ± 10.1, and 28.6 ± 9.6, respectively) and large oocytes groups (54.2 ± 13.5, 44.4 ± 3.9, and 67.6 ± 12.4, respectively). In addition, the probability of reaching the blastocyst stage was positively related to the total diameter ( p < 0.001), oocyte diameter ( p < 0.05), and ZP thickness ( p < 0.001). Furthermore, the relative gene expression of BAX , BCL2 , GDF9 , and GJA1 was lower in oocytes classified as large. In experiment 2, the mRNA transcript relative abundance pattern of genes in CCs was evaluated according to oocyte total diameter and developmental stage reached. CCs from oocytes classified as large and oocytes capable of developing to the blastocyst stage had a lower relative expression of BAX , STAR , and PTGS2 , while a higher expression of HAS2 and SDC2 transcript was observed for those oocytes. In conclusion, oocyte morphometric parameters and gene expression analysis in oocytes and CCs provide methods for the identification of the most competent oocytes for assisted reproductive technologies in sheep.

To date, the underlying mechanisms by which cAMP modulators act during in vitro maturation to improve oocyte developmental competence are poorly understood. Here, we sought to fill this knowledge gap by evaluating the use of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and adenylyl cyclase activator forskolin during a culture period of 2 h before in vitro maturation (pre-IVM) on the nuclear and cytoplasmic maturation features in essential organelles, cumulus cells activity, and in vitro developmental potential of sheep oocytes. Results showed that pre-IVM treatment significantly decreased ( p < 0.05) the DNA damage of mature oocytes (pre-IVM = 2.08% ± 3.51% vs. control = 20.58% ± 3.51%) and increased ( p ≤ 0.05) expanded blastocyst rates compared to the control (from the total of oocytes: pre-IVM = 23.89% ± 1.47% vs. control = 18.22% ± 1.47%, and from the cleaved embryos: pre-IVM = 45.16% ± 1.73% vs. control = 32.88% ± 1.73%). Considering that oocytes are highly vulnerable to the accumulation of DNA damage because of exposure to in vitro culture conditions, our results suggest that the modulation of intra-oocyte cAMP levels with forskolin and IBMX before IVM might afford oocytes a more effective DNA repair mechanism to overcome damage obstacles and ultimately improve developmental competence. This previously unappreciated action of cAMP modulators could help to develop improved methods for assisted reproduction technologies in animal and clinical research.

Knowledge of factors affecting semen quality could be of great importance for the collection and preservation of semen from threatened animals. To assess the effect of seasonality, sperm parameters and testosterone levels were examined throughout the year and compared with the distribution of conceptions. Cuvier's gazelle showed higher sperm quantity in April, coinciding with one peak of conceptions. In dorcas gazelle, sperm parameters showed a drop in October. However, percentage of conceptions increased during that month. In Mohor gazelle, sperm quality was best in April and August, in agreement with higher conception rates and high testosterone levels. Percentage of conceptions was correlated with photoperiod and rainfall in Cuvier's gazelle and with temperature in Mohor gazelle. To assess the effect of social environment, semen quality, testosterone and cortisol levels were quantified in males housed alone, in bachelor groups or with females. No differences were seen in Cuvier's and Mohor gazelles' semen traits, whereas dorcas males housed with females showed lower semen quality than males kept alone or with other males. Overall, ejaculate quality is influenced by seasonal factors in the three gazelle species, while social factors only appear to affect that of dorcas gazelle.

The aim of this study was to assess the effect of melatonin implants on the reproductive performance of yearling Iberian red deer ( Cervus elaphus hispanicus ) hinds. It also explored exogenous melatonin administration as a tool to minimize the negative effect of a low yearling hind's liveweight on their reproductive efficiency. In addition, the effect of melatonin-treated yearling hinds on non-treated hinds was studied in order to provide a practical and economical protocol to improve farms' productivity. A total of 4520 Iberian red deer hinds belonging to the same farm were included in this study. Melatonin (108 mg/hind) implants were administered three-fold every 30 days before the breeding season. Fertility rates, calves' weights and calving dates were registered for each hind. The results showed that exogenous melatonin increased significantly ( p < 0.05) the calves' weight (32.39 ± 1.07 kg vs. 27.65 ± 1.11 kg for Weight 1 calf (July) and 46.59 ± 1.50 kg vs. 41.79 ± 1.54 kg for Weight 2 calf (August, at weaning)) and advanced the calving date by 15 days in yearling hinds compared to the non-treated group. In addition, the administration of melatonin implants before the breeding season was able to minimize the negative effect of low yearling hinds' liveweight (Weight 1 hind ) on their future reproductive outcomes, as the fertility rates increased by 46% and the calves' weight increased by 7 kg after the melatonin treatment, regardless of the yearlings' weight. Finally, when both experimental groups (melatonin and non-treated) were kept separate, higher fertility rates (76.73 ± 7.18% vs. 66.94 ± 7.41%) were observed for the melatonin-treated hinds compared to the non-treated hinds. However, when both groups of yearling hinds were maintained together, no significant differences were observed in their fertility outcomes (78.13 ± 21.26% vs. 78.12 ± 23.32%). Therefore, melatonin implants may be used in yearling Iberian red deer hinds as a management tool to improve their reproductive productivity.

Background: Sperm chromatin structure provides valuable information for the prediction of male fertility and can be altered during different procedures. Previous studies have shown that sperm chromatin condensation decreased during in vitro capacitation. Moreover, cryopreservation can affect sperm DNA integrity and chromatin compaction., Objectives: This study aimed to investigate dynamic modifications produced in the chromatin structure of ram spermatozoa during in vitro capacitation before and after cryopreservation., Materials and Methods: Chromatin decondensation (AB+), DNA methylation, DNA fragmentation index (%DFI) and high DNA stainability (HDS) were evaluated in fresh and frozen-thawed ram spermatozoa incubated under capacitating (CAP) conditions at 1, 5, 15, 30, 60, 120, 180 and 240 minutes and under non-capacitating (NC) conditions at 0, 15 and 240 minutes., Results: Incubation in NC conditions did not induce significant changes in chromatin condensation (P > .05; AB + and HDS). However, incubation of fresh and cryopreserved ram spermatozoa under CAP conditions significantly increased chromatin decondensation (P < .05), reaching the highest percentage of AB + and HDS from 180 to 240 minutes in fresh samples and from 5 to 30 minutes in cryopreserved samples. Both variables (HDS and AB+) were positively correlated with tyrosine phosphorylation, total motility, progressive motility, curvilinear velocity and amplitude of lateral head displacement, as well as between them under CAP conditions in fresh and cryopreserved spermatozoa. DNA methylation significantly increased in cryopreserved spermatozoa (P < .05), but only after extended incubation under CAP conditions (60-240 minutes), while the %DFI, albeit higher in cryopreserved samples, remained constant under CAP and NC conditions in both types of sample (P > .05)., Discussion and Conclusions: Our results suggest that sperm chromatin condensation decreased progressively during in vitro capacitation of ram spermatozoa, while sperm DNA integrity remained intact. Such changes in chromatin condensation appeared faster after sperm cryopreservation., (© 2020 American Society of Andrology and European Academy of Andrology.)

For the past two decades, there has been a growing interest in the application of in vitro embryo production (IVP) in small ruminants such as sheep. To improve efficiency, a large number abattoir-derived ovaries must be used, and long distances from the laboratory are usually inevitable when adult animals are used. In that scenario, prolonged sheep ovary transportation may negatively affect oocyte developmental competence. Here, we evaluated the effect of ovary storage time (3, 5, 7, 9, 11 and 13 h) and the medium in which they were transported (TCM199 and saline solution) on oocyte quality. Thus, live/dead status, early apoptosis, DNA fragmentation, reduced glutathione (GSH) and reactive oxygen species (ROS) content, caspase-3 activity, mitochondrial membrane potential and distribution, and relative abundance of mRNA transcript levels were assessed in oocytes. After in vitro maturation (IVM), cumulus cell viability and quality, meiotic and fertilization competence, embryo rates and blastocyst quality were also evaluated. The results revealed that, after 7 h of storage, oocyte quality and developmental potential were significantly impaired since higher rates of dead oocytes and DNA fragmentation and lower rates of viable, matured and fertilized oocytes were observed. The percentage of cleavage, blastocyst rates and cumulus cell parameters (viability, active mitochondria and GSH/ROS ratio) were also decreased. Moreover, the preservation of ovaries in medium TCM199 had a detrimental effect on cumulus cells and oocyte competence. In conclusion, ovary transport times up to 5 h in saline solution are the most adequate storage conditions to maintain oocyte quality as well as developmental capacity in sheep. A strategy to rescue the poor developmental potential of stored oocytes will be necessary for successful production of high-quality embryos when longer ovarian preservation times are necessary.

Vitamin E is considered a powerful biological antioxidant; however, its characteristics such as high hydrophobicity and low stability limit its application. We propose to use nanotechnology as an innovative tool in spermatology, formulating nanoemulsions (NE) that accommodate vitamin E, protecting it from oxidation and promoting its release into the medium. The protective effect of the NE against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from eleven stags were thawed and extended to 400 × 10 6 sperm/ml in Bovine Gamete Medium (BGM). Once aliquoted, the samples were supplemented with the NE at different concentrations (0, 6 and 12 mM), with or without induced oxidative stress (100 μM Fe 2+ /ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. Motility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) were assessed. After 2 and 4 h of incubation, the NE were able to prevent the deleterious effects of oxidative stress, thus improving total and progression motility (P ˂0.05). Moreover, the highest concentration tested (12 mM) improved almost every sperm kinematic variable (P ˂0.05) and preserved sperm viability in samples subjected to oxidative stress. In addition, 12 mM of NE protected the acrosomes integrity, maintained and protected mitochondrial activity, prevented sperm lipoperoxidation and reduced ROS production (P ˂0.05) in samples subjected to oxidative stress. This work indicates for the first time that vitamin E formulated in NE could be a new approach against sperm oxidative damage. This could be highly relevant for sperm physiology preservation in the context of assisted reproduction techniques., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post-mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post-thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post-thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning., (© 2020 Blackwell Verlag GmbH.)

A major limiting factor for the development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, compared to livestock animals is the poor availability and limited access to biological material. Thus, the use of post-mortem ovaries from slaughtered animals represent a source of oocytes for the large scale production of embryos needed for research and to improve the efficiency of IVP. However, these oocytes are not as developmentally competent as their in vivo counterparts. Moreover, oocytes are usually obtained from ovaries that have been transported for long distances, which may also affect their quality. In order to overcome the issues associated with prolonged storage times of post-mortem material, in this study we examined the effect of melatonin supplementation to the ovary transport medium on oocyte quality, embryo yield, and blastocyst quality in Iberian red deer. When necessary, sheep was used as an experimental model due to the large number of samples required for analysis of oocyte quality parameters. Oocytes were in vitro matured and assessed for early apoptosis; DNA fragmentation; reactive oxygen species (ROS); reduced glutathione (GSH) content, mitochondrial membrane potential, and distribution; and relative abundance of mRNA transcript levels. After in vitro fertilization, embryo rates and blastocyst quality were also investigated. The results revealed that melatonin treatment significantly increased intracellular level of GSH in sheep oocytes. Moreover, the percentage of cleavage and blastocyst yield in red deer was greater compared to the Control group and there was lower abundance of oxidative stress- and apoptosis-related SHC1 , TP53 , and AKR1B1 mRNA transcripts in blastocysts for the Melatonin group. In conclusion, the supplementation of melatonin to the ovary storage medium had a positive effect on the developmental competence and quality of resulting blastocysts in Iberian red deer.

Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages ( p < 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater ( p < 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.

Sperm cryopreservation represents a powerful tool for livestock breeding. Several efforts have been made to improve the efficiency of sperm cryopreservation in different ruminant species. However, a significant amount of sperm still suffers considerable cryodamage, which may affect sperm quality and fertility. Recently, the use of different "omics" technologies in sperm cryobiology, especially proteomics studies, has led to a better understanding of the molecular modifications induced by sperm cryopreservation, facilitating the identification of different freezability biomarkers and certain proteins that can be added before cryopreservation to enhance sperm cryosurvival. This review provides an updated overview of the molecular mechanisms involved in sperm cryodamage, which are in part responsible for the structural, functional and fertility changes observed in frozen-thawed ruminant sperm. Moreover, the molecular basis of those factors that can affect the sperm freezing resilience of different ruminant species is also discussed as well as the molecular aspects of those novel strategies that have been developed to reduce sperm cryodamage, including new cryoprotectants, antioxidants, proteins, nanoparticles and vitrification.

The aim of this study was to investigate the dynamic changes that ram sperm experience during in vitro capacitation before and after cryopreservation. Using flow cytometry and computer assisted sperm analysis system (CASA), protein tyrosine phosphorylation and several functional parameters were evaluated in fresh and cryopreserved ram sperm incubated under capacitating and non-capacitating conditions at 0, 1, 5, 15, 30, 60, 120, 180 and 240 min. A short incubation period (5-30 min) under capacitating conditions was enough to increase mitochondrial activity and tyrosine phosphorylation in cryopreserved sperm, inducing also changes in the motility pattern, which could be related to hyperactivation. However, fresh sperm required a longer incubation (180-240 min) under capacitating conditions to undergo similar modifications. In both types of samples, tyrosine phosphorylation increased in a sequential manner in the midpiece, principal piece and tail at specific time points during in vitro capacitation. Moreover, the proportion of viable sperm with intact acrosome begun to decrease during capacitation, occurring before in cryopreserved sperm. Our findings suggest that cryopreserved ram sperm become competent for fertilization after a short exposure to capacitating conditions as a result of drastic changes inflicted by the freezing-thawing procedure, while prolonged incubations after cryopreservation severely impair sperm quality., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 μM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results., Competing Interests: Declaration of competing interest The authors have not declared any conflicts of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Incubation gas atmosphere affects the development of in vitro produced embryos. In this study, there was examination of effects of two different oxygen (O 2 ) tensions (5 % and 21 %) during in vitro maturation (M5 and M21) and/or fertilization (F5 and F21) on embryo production and quality in deer and sheep. There was assessment of the percentage of embryos with cell cleavage occurring, percentage that developed to the blastocyst stage, and analysis of the relative abundance of mRNA transcript for genes important for development to the blastocyst stage. The O 2 tension treatment did not affect (P > 0.05) percentage cleavage or blastocyst development in either species. In sheep, there was a greater abundance of SHC1, GPX1, TP53, BAX and NRF1 mRNA transcript (P < 0.05) in M21 F5-derived embryos. In deer, there was a greater abundance of SOD2 mRNA transcript (P < 0.05) when oocytes had been matured under relatively lesser O 2 , regardless of the tension used during fertilization. There was a lesser abundance of SOX2 mRNA transcript (P < 0.05) in the M5F21 compared to the other three treatment groups. The AKR1B1 mRNA transcript was in greater abundance (P < 0.05) in M21 F21 as compared to M21 F5 and M5F21 group, and there was a greater abundance PLAC8 mRNA transcript (P < 0.05) in M21 F21, as compared to all other treatment groups. In conclusion, while O 2 tension had no effect on developmental rates it did affect the relative abundance of mRNA transcript of multiple genes related to important cell functions during development., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nowadays, the use of foetal calf serum (FCS) during in vitro embryo culture is very controversial. Whilst some authors have encouraged its use, others reject it because of its harmful effects. Although in vitro embryo production in red deer is a promising assisted reproductive technique, it is still in its infancy and a great effort is needed to update the protocols used. The aim of this study was to assess whether FCS supplementation in red deer embryo culture medium is necessary to produce blastocyst and, if so, when is the best time to add it in terms of blastocyst production and quality. In vitro blastocysts were cultured with FCS added at 24, 48 or 96 hours post-insemination (hpi). In addition, a treatment without FCS was used as control. Six hundred and ninety-four cumulus-oocyte complexes were collected for in vitro fertilization. Cleavage rate was examined at 48 hpi, and blastocyst yield was recorded on days 6, 7 and 8. FCS had no influence on cleavage and blastocyst rate for any of the treatments studied. However, the number of cells was higher (p = .025) in those blastocysts cultured with FCS from 48 hpi compared with FCS-free culture media (93.88 ± 7.76 vs. 54.11 ± 8.36). In conclusion, the addition of FCS to the embryo culture medium at 48 hpi improves the quality of red deer blastocyst, although it does not affect the percentage of embryos obtained., (© 2019 Blackwell Verlag GmbH.)

Our aim was to optimize 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunodetection in order to detect DNA damage caused by oxidative stress that may not be detected by other DNA integrity analysis techniques, especially due to the high compaction of DNA in ruminants. Semen samples from 6 rams were cryopreserved. After thawing, samples were subjected to the DNA oxidation quantification using an 8-OHdG immunodetection assay by flow cytometry. We have evaluated two different incubation times (30 min vs. overnight) at 4°C of the primary antibody (monoclonal anti-8-OHdG antibody). We have also compared the results of this technique with the sperm chromatin structure assay (SCSA ® ). The analysis revealed that there were no significant differences (p > .05) between different incubation times. However, overnight incubation seems to cause more non-specific binding of the secondary antibody. Significant differences (p < .05) between subjects and oxidation controls (8 M H 2 O 2 /800 μM FeSO 4 •7H 2 O) were evident. We can conclude that the 8-OHdG immunodetection assay for DNA oxidation quantification of ram sperm can be performed subjecting sperm samples to a very high oxidative treatment., (© 2019 Blackwell Verlag GmbH.)

This study aimed to compare the ability of sperm chromatin structure assay (SCSA ® ) and Sperm-Ovis-Halomax ® to detect DNA fragmentation in frozen-thawed ram spermatozoa incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with oestrous sheep serum (SOF-ESS) at multiple time points (0-240 min). Incubation in SOF-ESS had no significant effects on SCSA ® parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm-Ovis-Halomax ® increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques, suggesting that SCSA ® and Sperm-Ovis-Halomax ® may quantify different types of DNA damage in ram spermatozoa under these experimental conditions., (© 2019 Blackwell Verlag GmbH.)

Mammalian sperm must undergo a set of structural and functional changes collectively termed as capacitation to ensure a successful oocyte fertilization. However, capacitation can be compromised by cryopreservation procedures, which alter the proteome and longevity of sperm. To date, how the protein changes induced by cryopreservation could affect the acquisition of sperm fertilizing potential remains unexplored. The present study investigated the protein profile of ram sperm during in vitro capacitation before and after cryopreservation to elucidate the impact of cryopreservation on sperm capacitation at a molecular level. Fresh and cryopreserved ram sperm were incubated under capacitating (CAP) and non-capacitating (NC) conditions for 240 min. The sperm proteome of these four treatments was analyzed and compared at different incubation times using reverse phase liquid chromatography coupled to mass spectrometry (RP-LC-MS/MS). The comparison between fresh and cryopreserved sperm suggested that cryopreservation facilitated an apoptosis-stress response and redox process, while the comparison between sperm incubated in CAP and NC conditions showed that capacitation increased those biological processes associated with signaling, metabolism, motility, and reproductive processes. In addition, 14 proteins related to mitochondrial activity, sperm motility, oocyte recognition, signaling, spermatogenesis, and the apoptosis-stress response underwent significant changes in abundance over time when fresh and cryopreserved sperm incubated in CAP and NC conditions were compared. Our results indicate that disturbances in a ram sperm proteome after cryopreservation may alter the quality of sperm and its specific machinery to sustain capacitation under in vitro conditions.

Cinnamtannin B-1 (CNB-1) is a naturally occurring trimeric A-type proanthocyanidin contained in several plants such as cinnamon (Cinnamomum zeylanicum). It is considered to be a potent antioxidant. The protective effect of CNB-1 against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from six stags were thawed, pooled and extended to 400 × 10 6 sperm/ml in BGM (bovine gamete medium). After being aliquoted, the samples were supplemented with different concentrations of CNB-1 (0, 0.1, 1, 10 and 100 μg/mL), with or without induced oxidative stress (100 μM Fe 2+ /ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. This experiment was replicated six times. Spermmotility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (TUNEL) were assessed. After 4 h of incubation, CNB-1 prevented the deleterious effects of oxidative stress, thus improved sperm progressivity and velocity (P<0.05). Furthermore, 1 and 10 μM CNB-1 improved sperm linearity, even when compared to those samples that had not been subjected to oxidative stress (P<0.05). The greatest concentration, 100 μM, prevented sperm lipoperoxidation and reduced ROS production in samples subjected to oxidative stress., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps ( P  < 0.0001), concomitantly with a similar percentage of fragmentized tracks ( P  = 0.0709) at sperm concentrations ranging 25-37 × 10 6  cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species., (© 2018 Society for Reproduction and Fertility.)

The aim of this study was to assess the effect of insemination timing on pregnancy rates in red deer (Cervus elaphus) when using sex-sorted sperm samples. Semen was collected by electroejaculation from 8 mature stags and processed to obtain: Conventional samples, following standard freezing procedures for commercial purposes; Control sorted samples, diluted and handled as per sorted samples but without being submitted to the sorter passage; and Y Sex Sorted (YSS) samples. Hinds were synchronized via intravaginal CIDR (Controlled Internal Drug Release) placement and given eCG (Folligon ® PMSG Serum Gonadotrophin) on day 12, upon CIDR removal. They were then inseminated with one of each sperm treatment, at the following post-eCG intervals: I&#95;1, 55:01-55:30 h; I&#95;2, 55:31-56:00 h; I&#95;3, 56:01-56:30 h; or, I&#95;4, 56:31-57:00 h. Pregnancy rates were assessed at parturition. Average pregnancy rates were highest (P < 0.05) for Conventional samples (77.6%), but similar between YSS (49.8%) and Control sorted (51.3%) samples. However, when insemination interval was taken into account, pregnancy rates within the YSS group, pregnancy rates were 80 and 83.1% for I&#95;1 and I&#95;2, respectively were obtained. Notably, these rates were similar (P > 0.05) to the average pregnancy rates obtained with Conventional samples (77.6%). As expected, YSS sperm yielded 94% male offspring contrasting with the 57% males obtained with Conventional and Control sorted samples. Our findings support the importance of developing specific insemination timing protocols to improve pregnancy rates when using frozen-thawed sex-sorted sperm. These findings provide the foundation for further investigations in order to determine why the YSS sperm are able to fertilize the oocyte in a shorter period of time than the conventional samples., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Currently, sperm reproductive biotechnologies such as sex sorting and cryopreservation are undoubtedly valuable tools for improving the economic and biological efficiency of red deer production systems. In this context, and because of the particular characteristics of this species (extensive exploitation typically far from laboratory facilities), a key goal is to optimize the design of an adequate handling protocol of sperm samples before samples are subjected to sex sorting and cryopreservation procedures to obtain better outputs from the application of these technologies. The main aim of this paper was to design an adequate protocol for Iberian red deer sperm handling before sex sorting by flow cytometry to obtain optimal yields when sex sorting is used in this species. Semen samples from 11 adult males were obtained by electroejaculation during the breeding season. In this study, we tested different protocols for the handling of Iberian red deer spermatozoa before sorting by using different concentrations of sperm (400 or 800 × 10 6 ) and adding or not Hoechst 33342 before the transport of samples to the sorting facilities. Based on the results, the most adequate method used to handle samples before sorting was transportation at a high sperm concentration (800 × 10 6 /mL) without Hoechst 33342. These transportation conditions in combination with Hoechst 33342 staining at 5.2 μL/mL once at the flow cytometry laboratory resulted in better (P < 0.05) sorting efficiency (99.9% of the samples showing split) than both, those samples transported at 400 × 10 6 sperm/mL (between 51.2 and 55.2% of the samples showing split) and those samples stained before transport at a sperm concentration of 400 × 10 6 sperm/mL (between 15.4 and 75.7% of the samples showing split). Sorting rates and sperm quality after sorting and cryopreservation was not affected (P > 0.05) by sperm handling before sorting. Moreover, the sorting yields were compatible with the practical application of these reproductive biotechnologies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The spermatozoon is the most diverse cell type known and this diversity is considered to reflect differences in sperm function. How the diversity in sperm morphology arose during speciation and what role the different specializations play in sperm function, however, remain incompletely characterized. This work reviews the hypotheses proposed to explain sperm morphological evolution, with a focus on some aspects of sperm morphometric evaluation; the ability of morphometrics to predict sperm cryoresistance and male fertility is also discussed. For this, the evaluation of patterns of change of sperm head morphometry throughout a process, instead of the study of the morphometric characteristics of the sperm head at different stages, allows a better identification of the males with different sperm cryoconservation ability. These new approaches, together with more studies employing a greater number of individuals, are needed to obtain novel results concerning the role of sperm morphometry on sperm function. Future studies should aim at understanding the causes of sperm design diversity and the mechanisms that generate them, giving increased attention to other sperm structures besides the sperm head. The implementation of scientific and technological advances could benefit the simultaneous examination of sperm phenotype and sperm function, demonstrating that sperm morphometry could be a useful tool for sperm assessment.

Semen collection for cryopreservation is a key step for small ruminant conservation programs. While in these species semen is mainly collected via artificial vagina (AV), electroejaculation (EE) provides a viable alternative for untrained males. Herein we investigated the effect of semen collection method on post-thaw sperm quality by comparing two small ruminant species, sheep and goats. Semen from Blanca-Celtibérica bucks and Manchega rams was collected by AV and EE on the same day and cryopreserved using a standard protocol. At thawing, sperm motion parameters were evaluated by CASA, whereas membrane stability (YO-PRO-1), sperm viability (propidium iodide, PI) and mitochondrial activity (Mitotracker Deep Red) were analyzed using flow cytometry. The semen collection method negatively affected post-thaw sperm quality in bucks but not in rams. Thus, in bucks, post-thaw sperm motility was higher for samples collected by AV as compared to those obtained via EE. Similarly, post-thaw sperm parameters evaluated by flow cytometry were worse for buck samples collected by EE than those collected by AV in the same species, or than ram samples regardless of collection method. These results suggest that ovine and caprine spermatozoa have a different response to the cryopreservation process depending upon the semen collection method used. We hypothesize that the EE procedure may lead to changes in the composition of the ejaculate in bucks that would make spermatozoa more susceptible to the cryopreservation process, whereas this procedure would have had no effect on ram spermatozoa. This assumption requires further investigation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage., (© 2016 The Author(s).)

Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3-7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1(+) and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase(-) spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Studies concerning the relationships between sperm size and velocity at the intraspecific level are quite limited and often yielded contradictory results across the animal kingdom. Intramale variation in sperm size may represent a meaningful factor to predict sperm velocity, due to its relationship with the level of sperm competition among related taxa. Because sperm phenotype is under post-copulatory sexual selection, we hypothesized that a reduced intramale variation in sperm size is associated with sperm competitiveness in red deer. Our results show that low variation in sperm size is strongly related to high sperm velocity and normal sperm morphology, which in turn are good predictors of male fertility in this species. Furthermore, it is well known that the red deer show high variability in testicular mass but there is limited knowledge concerning the significance of this phenomenon at intraspecific level, even though it may reveal interesting processes of sexual selection. Thereby, as a preliminary result, we found that absolute testes mass is negatively associated with intramale variation in sperm size. Our findings suggest that sperm size variation in red deer is under a strong selective force leading to increase sperm function efficiency, and reveal new insights into sexual selection mechanisms.

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter(2)/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r=-0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

The potential protective effect of reduced glutathione (GSH) and trolox (TRX), an analogue of vitamin E, supplementation during in vitro culture (2h, 39°C) of electroejaculated frozen/thawed red deer sperm was investigated. Cryopreserved sperm were thawed and incubated with no additive (Control) and 1mM or 5mM of each antioxidant to find out whether these supplementations can maintain the sperm quality, considering the use of thawed samples for in vitro techniques such as in vitro fertilisation (IVF), sperm sex sorting or refreezing. The effect of GSH on sperm motility was positive compared to TRX which was negative (P<0.001). After 2h of incubation at 39°C, use of GSH improved motility while TRX supplementation reduced sperm motility compared with Control samples without antioxidant. Use of TRX at both concentrations (1 and 5mM; TRX1 and TRX5) resulted in lesser percentages of apoptotic sperm (12.4±1.1% and 11.7±0.9%) than GSH1, GSH5 (15.2±1% and 14.6±1.1%) and Control samples (16.9±1.2%) (P<0.001). Use of GSH at both concentrations (1 and 5mM) resulted in greater mitochondrial activity as compared with findings for the Control, TRX1 and TRX5 groups. Results of this study indicate that GSH is a suitable supplement for electroejaculated red deer sperm. It would be necessary to conduct fertility trials (in vivo and in vitro), to assess whether GSH supplementation of thawed red deer sperm could improve fertility rates., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Spermatogenesis is a costly process that is expected to be under selection to maximise sperm quantity and quality. Testis size is often regarded as a proxy measure of sperm investment, implicitly overlooking the quantitative assessment of spermatogenesis. An enhanced understanding of testicular function, beyond testis size, may reveal further sexual traits involved in sperm quantity and quality. Here, we first estimated the inter-male variation in testicular function and sperm traits in red deer across the breeding and non-breeding seasons. Then, we analysed the relationships between the testis mass, eight parameters of spermatogenic function, and seven parameters of sperm quality. Our findings revealed that the Sertoli cell number and function parameters vary greatly between red deer males, and that spermatogenic activity co-varies with testis mass and sperm quality across the breeding and non-breeding seasons. For the first time in a seasonal breeder, we found that not only is the Sertoli cell number important in determining testis mass (r = 0.619, p = 0.007 and r = 0.248, p = 0.047 for the Sertoli cell number assessed by histology and cytology, respectively), but also sperm function (r = 0.703, p = 0.002 and r = 0.328, p = 0.012 for the Sertoli cell number assessed by histology and cytology, respectively). Testicular histology also revealed that a high Sertoli cell number per tubular cross-section is associated with high sperm production (r = 0.600, p = 0.009). Sperm production and function were also positively correlated (r = 0.384, p = 0.004), suggesting that these traits co-vary to maximise sperm fertilisation ability in red deer. In conclusion, our findings contribute to the understanding of the dynamics of spermatogenesis, and reveal new insights into the role of testicular function and the Sertoli cell number on testis size and sperm quality in red deer.

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin-hypotaurine alone, in combination with progesterone (P4), 17β-estradiol (E2), or BSA, or just β-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin-hypotaurine (alone), or in combination with P4, or E2, nor β-cyclodextrin induced capacitation-related changes in frozen-thawed ram spermatozoa. Only the medium supplemented with heparin-hypotaurine-BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin-hypotaurine, P4, E2, β-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The present study aimed to examine the behavior of ram spermatozoa subjected to a vitrification process in free-egg yolk diluents in relation with conventional diluents and cryopreservation protocol used in this species. Previously it was investigated the toxicity of cryoprotectants, sucrose and glycerol, based on different concentrations (sucrose at 0.03 M, 0.05 M, 0.15 M and 0.25 M; and glycerol at 3%, 7%, 14% and 18%) compared to a commercial extender (Biladyl® with 20% egg yolk and 7% glyerol). Cryoprotectants which reported less toxicity were chosen to perform the vitrification and results were compared with the conventional cryopreservation. Semen from three rams was collected by electroejaculation. The sperm evaluation was carried out at 0, 2 and 4h through the incubation time at 37°C for the experiment of toxicity and, at thawing when cryopreservation was performed. The sperm quality throughout the incubation time always resulted lower (P⩽0.05) for the free-egg yolk diluents in relation to Biladyl® (control), obtaining the lowest values of sperm quality with the highest concentrations of sucrose and glycerol. The vitrification was carried out with combinations of sucrose and glycerol (sucrose at 0.03 and 0.05 M with 3% and 7% of glycerol, respectively) and with Biladyl® (at different sperm concentrations). The vitrification decreased drastically (P⩽0.05) the sperm quality when combinations of sucrose and glycerol were used. Nevertheless, the sperm samples vitrified with Biladyl® at the lowest sperm concentration showed acceptable values of viability, acrosome integrity and DFI, although the sperm motility was strongly decreased. In conclusion, the use of vitrification with diluents based on combinations of sucrose and glycerol did not work for semen cryopreservation of ram. Promising results were obtained when diluents with egg yolk were used in the vitrification procedure, although more studies are necessary to improve this technique and the use of diluents without egg yolk., (Copyright © 2015 Elsevier Inc. All rights reserved.)