Lipid Enriched Reduced Nutrient Culture Medium Improves Bovine Blastocyst Formation.

The refinement of embryo culture media is essential in improving embryo viability and in vitro production efficiency. Our previous work demonstrated that the nutrients (carbohydrates, amino acids, and vitamins) in traditional culture media far exceed the need for an embryo and producing developmentally competent embryos in a reduced nutrient environment is feasible. Here, we aim to evaluate the impact of exogenous lipid and L-carnitine supplementation on bovine blastocyst development and refine our RN condition further. Zygotes were cultured in the control medium (100% nutrients) and reduced nutrient media containing 6.25% of the standard nutrient concentrations supplemented with L-carnitine and lipid free or lipid rich BSA. Increased blastocyst development was observed in the reduced nutrient lipid rich medium compared to the other two groups. However, in both reduced nutrient conditions, blastocyst cell numbers were lower than those obtained in the control condition. We then examined the expression level of 18 transcripts correlated with lipid metabolism, glucose metabolism, redox balance, and embryo quality, along with mitochondrial DNA copy numbers, ATP productions, and lipid profile. The results indicated lipid metabolism, embryo quality, and redox enzyme related genes were upregulated while glucose related gene was downregulated in embryos derived from reduced nutrient lipid rich condition Finally, we identified that the lipid rich BSA has enriched linoleic, stearic, oleic, palmitic, and alpha-linoleic fatty acids, a lipid profile that may contribute to the increased lipid metabolism and improved blastocyst development of the bovine embryos under the reduced nutrient condition.

Review: Reproductive physiology of bison and application of assisted reproductive technologies to their conservation.

Bison are an ecologically and culturally important species on the European and North American continents. Their near extirpation was met with conservation efforts that prevented their extinction but left few animals or highly fragmented populations. Contemporary conservation efforts are focused on building ecologically and genetically sustainable bison herds for long-term conservation of the species. Assisted reproductive technologies (ARTs) can play a key role in building these herds by facilitating the movement of genetics in the form of gametes and embryos, while protecting animal well-being and ensuring biosecurity of existing bison herds. In addition, ARTs such as gamete and embryo cryopreservation can be used to protect against future losses of genetic diversity through biobanking. In this review, a brief summary of basic bison reproductive physiology is presented followed by an overview of the current state of ART in Bison bison (American bison) and Bison bonasus (European bison or wisent). Research on ART ranging from artificial insemination to in vitro embryo transfer and cloning is discussed with particular regard to the application of ART for conservation purposes. While significant progress has been made in ART for bison, there are still many opportunities to improve these technologies and expand their impact for bison conservation.

Effect of cholesterol-loaded cyclodextrin treatment of bovine sperm on capacitation timing.

Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Cholesterol-loaded cyclodextrin (CLC) (2 mg/mL) improved post-thaw viability to 61% from 45% in control sperm (p < .05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p < .05). In conclusion, CLC-treated sperm required 1 h more to capacitate when compared with non-treated sperm based on percentage of live cells with high membrane disorder (p < .05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization.

Towards a method for cryopreservation of mosquito vectors of human pathogens.

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes.

A Reference Genome Assembly of American Bison, Bison bison bison.

Bison are an icon of the American West and an ecologically, commercially, and culturally important species. Despite numbering in the hundreds of thousands today, conservation concerns remain for the species, including the impact on genetic diversity of a severe bottleneck around the turn of the 20th century and genetic introgression from domestic cattle. Genetic diversity and admixture are best evaluated at genome-wide scale, for which a high-quality reference is necessary. Here, we use trio binning of long reads from a bison-Simmental cattle (Bos taurus taurus) male F1 hybrid to sequence and assemble the genome of the American plains bison (Bison bison bison). The male haplotype genome is chromosome-scale, with a total length of 2.65 Gb across 775 scaffolds (839 contigs) and a scaffold N50 of 87.8 Mb. Our bison genome is ~13× more contiguous overall and ~3400× more contiguous at the contig level than the current bison reference genome. The bison genome sequence presented here (ARS-UCSC_bison1.0) will enable new research into the evolutionary history of this iconic megafauna species and provide a new tool for the management of bison populations in federal and commercial herds.

A Reference Genome Assembly of Simmental Cattle, Bos taurus taurus.

Genomics research has relied principally on the establishment and curation of a reference genome for the species. However, it is increasingly recognized that a single reference genome cannot fully describe the extent of genetic variation within many widely distributed species. Pangenome representations are based on high-quality genome assemblies of multiple individuals and intended to represent the broadest possible diversity within a species. A Bovine Pangenome Consortium (BPC) has recently been established to begin assembling genomes from more than 600 recognized breeds of cattle, together with other related species to provide information on ancestral alleles and haplotypes. Previously reported de novo genome assemblies for Angus, Brahman, Hereford, and Highland breeds of cattle are part of the initial BPC effort. The present report describes a complete single haplotype assembly at chromosome-scale for a fullblood Simmental cow from an F1 bison-cattle hybrid fetus by trio binning. Simmental cattle, also known as Fleckvieh due to their red and white spots, originated in central Europe in the 1830s as a triple-purpose breed selected for draught, meat, and dairy production. There are over 50 million Simmental cattle in the world, known today for their fast growth and beef yields. This assembly (ARS_Simm1.0) is similar in length to the other bovine assemblies at 2.86 Gb, with a scaffold N50 of 102 Mb (max scaffold 156.8 Mb) and meets or exceeds the continuity of the best Bos taurus reference assemblies to date.

Production of embryos and a live offspring using post mortem reproductive material from bison (Bison bison bison) originating in Yellowstone National Park, USA.

Bison from Yellowstone National Park (YNP) have an important genetic history. As one of the few wild herds of bison with no evidence of cattle DNA introgression and a large enough population to maintain genetic diversity, they are considered a conservation priority for the species. Unfortunately, there is a high prevalence of the zoonotic disease brucellosis in the herd. Part of the management strategy for controlling the disease and herd size in YNP is to remove bison from the population during the winter migration out of the park. This interagency management cull provides an opportunity to collect a large number of oocytes from a wild bison population for genetic banking and research purposes. During the winters of 2014-2018, which is the nonbreeding season for bison, oocytes were collected post mortem and used to determine the effects of donor reproductive maturity and pregnancy status on oocyte quality and in vitro fertilization (IVF) outcomes, and to demonstrate the feasibility of producing healthy offspring. Cumulus oocyte complexes (COCs) were placed into an in vitro embryo production (IVP) system, and on days 7, 7.5, and 8 of in vitro culture (Day 0 = day of in vitro fertilization) embryos were assessed for developmental stage and quality prior to vitrification. Embryos were then stored in liquid nitrogen until the breeding season when a subset were warmed, cultured for 6 h, evaluated for survival, and transferred to healthy bison recipients. There were no significant differences in the ability of recovered COCs to support blastocyst development based on female reproductive maturity or pregnancy status (juvenile 79/959 (8.2%) vs sexually mature 547/6544 (8.4%); non-pregnant 188/2302 (8.2%) vs pregnant 556/6122 (9.1%)). Following the transfer of 15 embryos to 10 recipients, one healthy female calf was born. This work demonstrates that live offspring can be generated from COCs collected from YNP bison post mortem in the non-breeding season, and that gamete recovery can be a valuable tool for conservation of valuable genetics for this species while mitigating diseases like brucellosis.

In Vitro Production of Bison Embryos.

Bison are an iconic species of cultural, conservation, and commercial interest. Various assisted reproductive technologies have been tested in bison over the last few decades (e.g., superovulation and embryo transfer), but their efficiencies are low. Since 2007, several methods for producing bison embryos in vitro have been published. All of these methods are based on cattle embryo production models and have varying degrees of success with regard to embryo production rates. In this chapter, a brief summary of these reports is presented followed by a detailed protocol that has been successfully used to produce bison embryos in vitro and live offspring following embryo transfer.

Alterations in oocyte mitochondrial number and function are related to spindle defects and occur with maternal aging in mice and humans†.

The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility.

Transporting cumulus complexes using novel meiotic arresting conditions permits maintenance of oocyte developmental competence.

PURPOSE: The aim of this study is to evaluate the effect of a novel bovine cumulus oocyte complex (COC) shipping media designed to arrest meiotic resumption during transport on meiotic arrest, as well as meiotic resumption, subsequent embryonic development, and embryo quality. METHODS: Bovine cumulus oocyte complexes were transported overnight from the collection facility to the laboratory. COCs were placed in control in vitro maturation (IVM) or in shipping arrest medium (SAM) containing multiple meiotic inhibitors, and then shipped to our laboratory. Upon arrival, meiotic status was assessed, control COCs were inseminated, and arrested COCs were matured and inseminated the next day. Embryonic development and quality were analyzed. RESULTS: When bovine COC arrived at the laboratory after overnight shipment (21 h) in SAM, the majority of oocytes remained at the GV stage (75.6 ± 2.9% GV). Arrested oocytes successfully resumed and completed meiosis during IVM after removal from SAM (96.8 ± 0.5% metaphase II compared to control 88.3 ± 5.0%). Moreover, the development of blastocysts per COC was not different from control (22.3 ± 2.4% for control and 18.7 ± 2.1% for SAM), nor was any difference detected in blastocyst quality as determined by cell number and allocation. CONCLUSIONS: Our study demonstrates that a physiological system incorporating cyclic adenosine monophosphate and cyclic guanosine monophosphate modulators can be used to maintain meiotic arrest followed by successful nuclear maturation and pre-implantation embryo development equal to control IVM-derived embryos. Our results offer promising insights for the development of pre-IVM media that may improve oocyte developmental competence in vitro.

A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

STUDY QUESTION: Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? SUMMARY ANSWER: A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. WHAT IS KNOWN ALREADY: Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 ± 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 ± 7.4% versus 53.3 ± 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 ± 9%, 37.2 ± 9.5% and 17.2 ± 8.3%, respectively), although fetal development did not differ. LIMITATIONS, REASONS FOR CAUTION: Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. WIDER IMPLICATIONS OF THE FINDINGS: IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. LARGE SCALE DATA: N/A. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.

Cryopreservation of bison epididymal sperm: A strategy for improving post-thaw quality when collecting sperm in field conditions.

This study was conducted to optimize the cryopreservation of epididymal bison sperm harvested in the field. In the first experiment, epididymal bison sperm were treated with or without seminal plasma (n = 6) and cooled to 5 °C over 2 hours. In a separate experiment, glycerol was added at different times and sperm was held at 5 °C for different periods of time before cryopreservation (n = 11). In addition, epididymal sperm frozen with and without seminal plasma (n = 6) and after 4, 24, and 48 hours (n = 5) of equilibration at 5 °C, were evaluated for their in vitro fertilizing ability. Post-thaw motility of bison epididymal sperm was similar when cryopreserved with or without seminal plasma or when glycerol was added at either 0, 4, 24, or 48 hours before freezing (P > 0.05). However, sperm incubated at 5 °C for 24 hours before freezing exhibited higher percentages of motile sperm (44% vs. 35% for 4 hours or 48 hours, P < 0.05). Fertilization rates of bison oocytes were not different for any treatments. Chilling the whole epididymis for 24 or 48 hours resulted in complete loss of sperm viability. In conclusion, bison epididymal sperm can be chilled outside of the epididymis for at least 48 hours before cryopreservation without compromising post-thaw sperm motility providing flexibility for technicians performing field collections.

Utility of infertile male models for contraception and conservation.

Epididymal defects in infertile domestic species and transgenic mice demonstrate the role of the epididymis in influencing sperm function. Spermatozoa from these males cannot negotiate the female tract as they fail to regulate their volume. The latter is necessary to counter the osmotic stresses encountered in the female tract. Reduced epididymal provision of osmolytes or their premature loss is discussed as probable causes of failed volume regulation. Measuring cell volume regulation for diagnosis of male infertility and blocking it as a means to male contraception are briefly considered. Unchecked human population growth is destroying habitats supporting vulnerable and endangered species. Genome resource banks have been established to preserve spermatozoa of genetically valuable individuals. As cryopreservation stresses spermatozoa osmotically, this process could jeopardise volume regulation with consequences for fertility. Knowledge of sperm volume regulation and the uptake of organic solutes may permit improvement in sperm storage and prevent osmolyte-related cryodamage.

Fertility control in wildlife: humans as a model.

In a time of accelerated extinction rates and biodiversity loss, it may seem illogical to be concerned with methods limiting the reproduction of wild populations; however, there is an urgent need to inhibit the proliferation of a wide variety of species. The range of animals for which fertility control is desired makes the development of a single method impossible. The various reproductive strategies used by individual species, the desired outcome of contraceptive programs (reversible or irreversible; male or female directed) and our ignorance of the reproductive biology of many endangered species necessitate thorough species-specific investigations. As fertility control in humans is a reality and research into methods of fertility control is more advanced, humans serve as a model for developing contraceptive approaches for wild species. Population control by traditional methods (indirect or direct intervention by culling, poisoning, translocation, etc.) is increasingly unacceptable to the public, making human studies even more valuable for finding solutions to overabundant wild populations. This review compares and contrasts the range of contraceptive methods used in both wildlife and humans.

The role of anion channels and Ca2+ in addition to K+ channels in the physiological volume regulation of murine spermatozoa.

Studies in the human, transgenic mice, and cattle indicate that sperm cell volume regulation plays an important role in male fertility as spermatozoa encounter a hypo-osmotic challenge upon ejaculation into the female tract. Physiological regulatory volume decrease (RVD) was examined using flow cytometry in murine sperm released into incubation medium mimicking uterine osmolality and including putative channel inhibitors. The involvement of K+ channels was indicated by the recovery of volume regulation by the K+ ionophore valinomycin in defective sperm from infertile transgenic mice, and from blockage of RVD by quinine in normal sperm. However, in neither case was the recovery complete. The involvement of volume-sensitive osmolyte and anion channels (VSOAC) were investigated using blockers effective in other cell types. NPPB (5-nitro-2(3-phenylpropylamino) benzoic acid) and tamoxifen inhibited RVD but SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid) at 0.4 and 1 mM had no effect whereas DIDS (di-isothiocyanato-stilbene-2,2'-disulphonic acid) at 1 mM enhanced RVD. Verapamil, but not another P-glycoprotein antagonist cyclosporin, caused sperm swelling which persisted in the presence of valinomycin, in Ca2+-free medium and in the presence of thapsigargin, but swelling was abolished by the Ca2+ ionophore A23187. Nifedipine was slightly effective in blocking RVD. Analysis by Western blotting failed to reveal ClC-2 and ClC-3 members of the chloride channel family in murine or rat sperm proteins despite signal bands in positive tissue controls. These findings implicate the involvement of some unidentified VSOAC in sperm volume regulation, which is probably Ca+-dependent.

Changes in osmolality during liquefaction of human semen.

The osmolality of 18 liquefying human semen samples from 15 volunteers was measured by vapour pressure osmometry to be low (294 mmol/kg, range 269-311). For each sample the osmolality increased during liquefaction to reach a mean of 312 mmol/kg (280-331) by 30 min at 37 degrees C. These results are at variance with the widely held view that semen osmolality is greater than that of serum, which results from its first being examined after liquefaction in vitro. Thus when sperm are routinely examined after liquefaction they have been subjected to osmotic stresses that are not experienced by spermatozoa entering the female tract at coitus.