Ice formation and its elimination in cryopreservation of oocytes.

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming were examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions - consistent with crystallization of most free water - during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

The evolution of centriole degradation in mouse sperm.

Centrioles are subcellular organelles found at the cilia base with an evolutionarily conserved structure and a shock absorber-like function. In sperm, centrioles are found at the flagellum base and are essential for embryo development in basal animals. Yet, sperm centrioles have evolved diverse forms, sometimes acting like a transmission system, as in cattle, and sometimes becoming dispensable, as in house mice. How the essential sperm centriole evolved to become dispensable in some organisms is unclear. Here, we test the hypothesis that this transition occurred through a cascade of evolutionary changes to the proteins, structure, and function of sperm centrioles and was possibly driven by sperm competition. We found that the final steps in this cascade are associated with a change in the primary structure of the centriolar inner scaffold protein FAM161A in rodents. This information provides the first insight into the molecular mechanisms and adaptive evolution underlying a major evolutionary transition within the internal structure of the mammalian sperm neck.

Inflammatory markers for differentiation of endometritis in the mare.

BACKGROUND: Endometrial biopsy is required to diagnose mares with chronic endometritis and endometrial degenerative fibrosis. An increase in understanding of equine reproductive immunology could be utilised to create less-invasive, time-efficient diagnostic tools especially when evaluating mares for chronic endometritis. OBJECTIVES: To evaluate inflammatory cytokine and chemokine concentrations in uterine fluid samples collected by low-volume lavage (LVL) as a potential screening diagnostic biomarker for endometritis. STUDY DESIGN: Prospective cross-sectional clinical study. METHODS: Forty-six mares underwent a LVL and subsequently endometrial biopsy. Mares were split in three groups: healthy, acute endometritis, and chronic endometrial fibrosis (CEF) based on cytological and histological evaluation. A fluorescent bead-based multiplex assay for IFN-γ, IFN-α, IL-1ß, IL-4, IL-10, IL-17, sCD14, TNF-α, CCL2, CCL3, CCL5 and CCL11 were carried out on the LVL fluid. The endometrial biopsy was utilised for histology and qPCR of IFN-γ, IL-1ß, IL-6, IL-8, IL-17, TNF-α, CCL2 and CCL3 genes. Statistical analyses examined differences in inflammatory markers and predictive modelling for diseased endometrium. RESULTS: Secreted concentrations of IFN-γ were lower in LVL fluid from reproductively healthy mares compared with acute endometritis (p = 0.04) and CEF (p = 0.006). Additionally, IL-17, IL-10, IL-1ß, TNF-α, CCL2, CCL3, CCL5 and CCL11 were significantly increased (p ≤ 0.04) in LVL from CEF mares compared with healthy mares. Mares with CCL2 concentrations ≥550 pg/mL (14/14) had 100% probability of having CEF and/or acute endometritis. Healthy mares had lower relative abundance of IL-17 mRNA compared with mares in CEF group [median (interquartile rage) = 14.76 (13.3, 15.3) and 12.4 (10.54, 13.81)], respectively (p = 0.02). MAIN LIMITATIONS: Limited sample size: larger numbers of mares with and without endometritis are required and reference intervals in LVL samples have to be established. CONCLUSIONS: Inflammatory chemokines and cytokines concentrations differed between healthy mares and mares with acute endometritis or CEF in LVL.

Faster sperm selected by rheotaxis leads to superior early embryonic development in vitro.

To understand the impact of sperm speed as they swim against the flow on fertilization rates, we created conditions similar to the female reproductive tract (FRT) on a microfluidic platform for sperm selection. Selected sperm were evaluated based on early development of fertilized embryos. Bovine and human spermatozoa were selected at various fluid flow rates within the device. We found that the speed of bovine spermatozoa increases as the flow rate increases and that the amount of DNA fragmentation index is lowered by increasing the flow rate. Bovine spermatozoa selected by our platform at low (150 µL h-1, shear rate 3 s-1), medium (250 µL h-1, shear rate 5 s-1), and high flow rates (350 µL h-1, shear rate 7 s-1) were used for fertilization and compared to sperm sorted by centrifugation. The samples collected at the highest flow rate resulted in the formation of 23% more blastocysts compared to the control. While selecting for higher quality sperm by increasing the flow rate does result in lower sperm yield, quality improvement and yield may be balanced by better embryonic development.

The uterine secretory cycle: recurring physiology of endometrial outputs that setup the uterine luminal microenvironment.

Conserved in female reproduction across all mammalian species is the estrous cycle and its regulation by the hypothalamic-pituitary-gonadal (HPG) axis, a collective of intersected hormonal events that are crucial for ensuring uterine fertility. Nonetheless, knowledge of the direct mediators that synchronously shape the uterine microenvironment for successive yet distinct events, such as the transit of sperm and support for progressive stages of preimplantation embryo development, remain principally deficient. Toward understanding the timed endometrial outputs that permit luminal events as directed by the estrous cycle, we used Bovidae as a model system to uniquely surface sample and study temporal shifts to in vivo endometrial transcripts that encode for proteins destined to be secreted. The results revealed the full quantitative profile of endometrial components that shape the uterine luminal microenvironment at distinct phases of the estrous cycle (estrus, metestrus, diestrus, and proestrus). In interpreting this comprehensive log of stage-specific endometrial secretions, we define the "uterine secretory cycle" and extract a predictive understanding of recurring physiological actions regulated within the uterine lumen in anticipation of sperm and preimplantation embryonic stages. This repetitive microenvironmental preparedness to sequentially provide operative support was a stable intrinsic framework, with only limited responses to sperm or embryos if encountered in the lumen within the cyclic time period. In uncovering the secretory cycle and unraveling realistic biological processes, we present novel foundational knowledge of terminal effectors controlled by the HPG axis to direct a recurring sequence of vital functions within the uterine lumen.NEW & NOTEWORTHY This study unravels the recurring sequence of changes within the uterus that supports vital functions (sperm transit and development of preimplantation embryonic stages) during the reproductive cycle in female Ruminantia. These data present new systems knowledge in uterine reproductive physiology crucial for setting up in vitro biomimicry and artificial environments for assisted reproduction technologies for a range of mammalian species.

Ice formation and its elimination in cryopreservation of bovine oocytes.

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction using cryopreserved oocytes and embryos. We use synchrotron-based time-resolved x-ray diffraction and tools from protein cryocrystallography to characterize ice formation within bovine oocytes after cooling at rates between ∼1000 °C/min and ∼600,000°C /min and during warming at rates between 20,000 and 150,000 °C /min. Maximum crystalline ice diffraction intensity, maximum ice volume, and maximum ice grain size are always observed during warming. All decrease with increasing CPA concentration, consistent with the decreasing free water fraction. With the cooling rates, warming rates and CPA concentrations of current practice, oocytes may show no ice after cooling but always develop substantial ice fractions on warming, and modestly reducing CPA concentrations causes substantial ice to form during cooling. With much larger cooling and warming rates achieved using cryocrystallography tools, oocytes soaked as in current practice remain essentially ice free during both cooling and warming, and when soaked in half-strength CPA solution oocytes remain ice free after cooling and develop small grain ice during warming. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes, establish the character of ice formed, and suggest that substantial further improvements in warming rates are feasible. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development, allowing other deleterious effects of the cryopreservation cycle to be studied, and osmotic stress and CPA toxicity reduced. Significance Statement: Cryopreservation of oocytes and embryos is critical in assisted reproduction of humans and domestic animals and in preservation of endangered species. Success rates are limited by damage from crystalline ice, toxicity of cryoprotective agents (CPAs), and damage from osmotic stress. Time-resolved x-ray diffraction of bovine oocytes shows that ice forms much more readily during warming than during cooling, that maximum ice fractions always occur during warming, and that the tools and large CPA concentrations of current protocols can at best only prevent ice formation during cooling. Using tools from cryocrystallography that give dramatically larger cooling and warming rates, ice formation can be completely eliminated and required CPA concentrations substantially reduced, expanding the scope for species-specific optimization of post-thaw reproductive outcomes.

Rho-associated coiled-coil containing kinase inhibitor improves outcomes of direct-transfer slow-cooled bovine blastocysts.

Direct-transfer slow-cooling cryopreservation is a widely used method for bovine embryo cryopreservation. However, the transfer of cryopreserved embryos is associated with reduced pregnancy rates. Rho-associated coiled-coil containing kinase inhibitor (ROCKi) has shown promise in improving the viability of post-warmed vitrified bovine embryos. Our objective was to investigate the effects of ROCKi treatment prior to slow-cooling or after cryopreservation on embryo viability. In vitro produced bovine embryos (n = 571) were randomly assigned to one of five groups: No-cryopreservation control group (NC-C), C-C group were cryopreserved by slow-rate cooling without ROCKi at any point, R-C group were incubated with ROCKi for 2 h before cryopreservation, C-R group were not exposed to ROCKi prior to cryopreservation but were cultured with ROCKi after cryopreservation, and R-R group were exposed to ROCKi before and after cryopreservation. Treatment group was significantly associated with blastocoel re-expansion, hatching, and degeneration (P < 0.0001). Blastocoel re-expansion rates were lower (P < 0.05) in the C-C (75.2 ± 4.2%) and R-C (85.2 ± 4.7%) groups compared with the NC-C (99.0 ± 0.7%), C-R (94.7 ± 2.6%) and the R-R (94.5 ± 2.9%) groups. The median time to re-expansion was significantly slowest in the C-C group (650, 560-915 min), followed by the R-C group (538, 421-611 min), then the C-R and R-R groups were similar (291, 261-361 and 321, 271-371 min) and the NC-C group was the fastest (196, 161-230 min) (P < 0.05). Similarly, the post-thaw hatching rate was lower, and the median time to hatching slower in the C-C (58.1 ± 7.0%, 2,033, 1634-2820 min) and R-C (65.7 ± 6.9%, 1,853, 1494-2356 min) groups compared with the NC-C (81.7 ± 6.0%, 1,309, 1084-1514 min), C-R (77.2 ± 6.5%, 1,384, 1013-1754 min) and R-R (82.0 ± 5.3%, 1,209, 943-1424 min) groups. ROCKi supplementation after cryopreservation resulted in fewer degenerated embryos (C-R = 8.9 ± 2.8%, and R-R 7.1 ± 2.8%) compared to the C-C (26.8 ± 4.3%) and R-C (17.9 ± 5.7%) groups. Exposure to ROCKi both before cryopreservation and after-cryopreservation yielded the best outcomes, similar to NC-C control group without cryopreservation, and significantly better than the C-C control group without supplements. Exposure to ROCKi after cryopreservation demonstrated greater benefits compared to exposure before cryopreservation alone. These findings suggest that ROCKi can potentially enhance cryosurvival of bovine embryos.

Canine intrahepatic portosystemic shunts: Interlobar and intralobar classifications.

This two-part study design showed that a canine congenital intrahepatic portosystemic shunt (IPSS) may be classified by its location within a liver fissure (interlobar) or lobe (intralobar). A prospective anatomic study reviewed normal canine liver morphology and showed the CT angiography (CTA) appearance of the normal canine ductus venosus (DV), which was confirmed via dissection and literature review to be between the papillary process and left-lateral liver lobe (in the fissure for ligamentum venosum). A retrospective multi-institutional case series documented the frequency of imaging findings in 56 dogs with a single IPSS that underwent portal CTA at Cornell University or the Schwarzman Animal Medical Center between June 2008 and August 2022. An interlobar IPSS was seen in 24 of 56 (43%) dogs, all arose from the left portal branch except one. These shunts were typically near the median plane, remained interlobar throughout the course, and were nearly always (96%) craniodorsal to the porta hepatis. Four types were distinguished: patent DV (11 dogs), left interlobar (11 dogs), right interlobar (1 dog), and ventral interlobar (1 dog). Only about half (46%) were in the fissure for ligamentum venosum and therefore classified as a patent DV. An intralobar IPSS was seen in 32 of 56 (57%) dogs, most (88%) originated from the right portal branch and were in the right-lateral liver lobe (21 dogs) or caudate process (7 dogs). During canine portal CTA, documenting the interlobar or intralobar location of an IPSS might increase the consistency and validity of IPSS description.

Extreme plasticity of reproductive state in a female rodent.

Successful sexual reproduction relies on the coordination of multiple biological systems, yet traditional concepts of biological sex often ignore the natural plasticity in morphology and physiology underlying sex. Most female mammals develop a patent (i.e., opened) vaginal entrance (introitus) prenatally or postnatally before or during puberty, usually under the influence of estrogens, and remain patent for the remainder of their lifespan1. An exception is the southern African giant pouched rat (Cricetomys ansorgei), whose vaginal introitus remains sealed well into adulthood2. Here, we explore this phenomenon and report that the reproductive organs and the vaginal introitus can undergo astounding and reversible transformation. Non-patency is characterized by reduced uterine size and the presence of a sealed vaginal introitus. Furthermore, the female urine metabolome shows that patent and non-patent females profoundly differ in their urine content, a reflection of differences in physiology and metabolism. Surprisingly, patency state did not predict fecal estradiol or progesterone metabolite concentrations. Exploring the plasticity that exists in reproductive anatomy and physiology can uncover that traits long considered 'fixed' in adulthood can become plastic under specific evolutionary pressures. Moreover, the barriers to reproduction that such plasticity creates present unique challenges to maximizing reproductive potential.

First Kiso pony foal produced via transfer of long-distance shipped fresh embryo to Hokkaido native pony.

Japanese native horses, which consists of 8 breeds, are threatened with extinction. Embryo transfer (ET) is used to reproduce endangered animals in various mammalian species. We aimed to perform ET using native ponies from Kiso and Hokkaido as donors and recipients, respectively. ET operation included long-distance transport of non-cryopreserved embryos from Nagano Prefecture to Hokkaido. Embryos were transported 1500 km over 9 h in a container maintained at 22°C. After transferring two embryos to two recipients, one mare delivered a healthy live foal. These results demonstrated that reciprocal ET with long-distance transportation of fresh embryos between the isolated breeds may allow for the proliferation of Japanese native horses.

Transient increases in glutamate dehydrogenase (GLDH) activity occur in neonatal foals.

BACKGROUND: Liver analyte measurement is important in the evaluation of sick animals. Liver injury in horses is recognized by increased glutamate dehydrogenase (GLDH), sorbitol dehydrogenase (SDH), and aspartate aminotransferase (AST) activities, whereas biliary pathology is identified by increased alkaline phosphatase and γ-glutamyl transferase (GGT) activities or bilirubin concentrations. We have observed high GLDH, but not SDH, activities in neonatal foals admitted for conditions other than liver disease. Only one previous study have evaluated GLDH activity over time in healthy neonatal foals; however, SDH activity was not measured. OBJECTIVE: We aimed to evaluate changes in liver analytes in neonatal foals over time. METHODS: We measured serum liver analytes (GLDH, SDH, GGT, AST, total, direct, and indirect bilirubin) and creatine kinase activity of 11 clinically healthy foals before and at various times after suckling until 46 days of age. Analytes were also measured in colostrum and mare serum. RESULTS: Median GLDH activities increased after birth to peak at 3-4 days of age (106 U/L, reference interval, 0-8 U/L). Median SDH activities had a lower peak at 3-4 days (15 U/L, reference interval, 0-11 U/L) and were frequently discordant with GLDH. There was no association between foal and mare serum or colostral enzyme activities. AST activity plateaued at 5-6 days, whereas GGT activity and total and indirect bilirubin concentrations peaked at 14 and 3-4 days of age, respectively. CONCLUSIONS: Transient increases in GLDH, SDH, and GGT activities and total and indirect bilirubin concentrations occur in clinically healthy neonatal foals and do not necessarily indicate relevant liver disease.

RhoA/ROCK signaling antagonizes bovine trophoblast stem cell self-renewal and regulates preimplantation embryo size and differentiation.

Exponential proliferation of trophoblast stem cells (TSC) is crucial in Ruminantia to maximize numerical access to caruncles, the restricted uterine sites that permit implantation. When translating systems biology of the undifferentiated bovine trophectoderm, we uncovered that inhibition of RhoA/Rock promoted self-renewing proliferation and substantially increased blastocyst size. Analysis of transcripts suppressed by Rock inhibition revealed transforming growth factor ß1 (TGFß1) as a primary upstream effector. TGFß1 treatment induced changes consistent with differentiation in bTSCs, a response that could be replicated by induced expression of the bovine ROCK2 transgene. Rocki could partially antagonize TGFß1 effects, and TGFß receptor inhibition promoted proliferation identical to Rocki, indicating an all-encompassing upstream regulation. Morphological differentiation included formation of binucleate cells and infrequent multinucleate syncytia, features we also localize in the in vivo bovine placenta. Collectively, we demonstrate a central role for TGFß1, RhoA and Rock in inducing bTSC differentiation, attenuation of which is sufficient to sustain self-renewal and proliferation linked to blastocyst size and preimplantation development. Unraveling these mechanisms augments evolutionary/comparative physiology of the trophoblast cell lineage and placental development in eutherians.

Incidence and risk factors for canine mastitis and metritis in two guide dog populations.

Canine mastitis and metritis can cause severe illness but the incidence and risk factors have not been well-studied. Goals in the present study were: 1) report the incidence of mastitis and metritis in a large population, and 2) identify potential risk factors that predispose females to those diseases. A retrospective cohort study was conducted using data from two guide dog colonies that was collected for 17 and 10 years, respectively, for the two colonies. A total of 3076 whelpings occurred during the respective study periods and data were analyzed. Clinical mastitis was diagnosed in 13.2 % of whelpings (408 cases) with the average day of diagnosis being 16.7 postpartum. Risk factors for mastitis identified were colony, litter size where bitches that had large litter size of ≥9 pups (16.2 %) were 60 % more likely to develop mastitis compared with bitches that had litters of <9 pups (11.3 %). Bitches with congestion of the mammary gland were 4.8 times more likely to develop mastitis compared with bitches without mammary congestion. Case incidence of metritis was small (0.7 % of whelpings) and occurred on average at day-5 postpartum (range 1-16). There were no significant risk factors identified, and this may be due to the small number of metritis cases (22 cases) in the present study. Interpretations regarding metritis, therefore, should be made with caution. The results from this study provide parameters for breeders and veterinarians to identify bitches that may require close monitoring for mastitis and metritis.

Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos.

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm300 µm). However, more research is required to find the best method for embryos ≥700 µm.

Malunion of an In Utero Metacarpal Fracture in a Standardbred Mare Presenting for Dystocia.

In utero fracture and malunion of long bones is a rare condition in horses. Most foals with in utero fractures are aborted, and the identification of a fetal in utero fracture in a mare with dystocia has not been reported. A 7-year-old multiparous Standardbred mare presented to a referral center for correction of dystocia. Assisted vaginal delivery and controlled vaginal delivery attempts were unsuccessful mainly because of contracted tendons impeding mutation. As the foal was alive, a cesarean section was elected. The foal was delivered but ultimately euthanized because of the congenital abnormalities. Computed tomography of the right forelimb of the foal along with gross examination and histologic evaluation of the right metacarpus revealed the malunion of a previous in utero fracture. While a few cases have been reported of in utero fracture, many of these were in abortuses and not in fetuses at term, making this case a new presentation and potential etiology for dystocia.

Uterine perforation secondary to metritis and placenta percreta in a postpartum bitch.

A 3-year-old intact female Labradoodle bitch was referred due to fever and lethargy 4 days postpartum. The dog was reported to have had prolonged labor that required assistance and fetal membranes were retained. Physical examination and diagnostics led to a suspicion of metritis and uterine perforation. Ovariohysterectomy was performed. Gross and histopathology findings revealed multifocal uterine perforation, necrosuppurative metritis, and placenta percreta. Post-operative antibiotic therapy and supportive care resulted in an uneventful clinical recovery. This is the first reported case of placenta percreta in a bitch. It is presumed that this pathology was paramount in the patient's development of metritis and subsequent uterine rupture. Key clinical message: Placenta percreta may lead to more severe clinical consequences of metritis, including uterine rupture.

Perforation utérine secondaire à une métrite et un placenta percreta chez une chienne en période post-partum. Une femelle Labradoodle intacte âgée de 3 ans fut référée pour cause de fièvre et léthargie 4 jours post-partum. Il fut rapporté que la chienne avait eu un travail long qui demanda de l'assistance et qu'il y avait eu rétention des membranes foetales. L'examen physique et le diagnostic mena à un doute de métrite et de perforation utérine. Une ovario-hystérectomie fut réalisée. Les trouvailles de pathologie macroscopique et d'histopathologie révélèrent des perforations utérines multifocales, une métrite nécro-suppurative et un placenta percreta. Une antibiothérapie post-opératoire et des soins de support ont résulté en une guérison clinique sans conséquence. Ceci représente le premier cas rapporté de placenta percreta chez une chienne. Il est présumé que chez cette chienne cette pathologie était vitale dans le développement de la métrite et de la rupture utérine subséquente.Message clinique clé :Un placenta percreta peut mener à des conséquences cliniques plus sévères de métrite, incluant la rupture utérine.(Traduit par Dr Serge Messier).

Colostral and foal serum immunoglobulin G levels and associations with perinatal abnormalities in heavy draft horses in Japan.

The purpose of this study was to elucidate the colostral and foal serum immunoglobulin G (IgG) concentration values in heavy draft horses in Japan and to examine the effects of peripartum mare condition on colostral immunity. Colostrum was obtained 1 hr after foaling (pre-suckling; n=178). Blood was collected from the jugular vein of the foals (n=147) at 24 to 48 hr after birth. The foaling statuses of 73 mares were recorded. The average colostral IgG concentration was 10,540 ± 3,190 mg/dl (median=10,928; range 1,434-17,514 mg/dl). The average serum IgG concentration obtained from neonatal foals 24 to 48 hr after birth was 1,750 ± 919 mg/dl (median=1,890; range 0-3,510 mg/dl). Although colostral IgG did not differ between the normal foaling mare (n=59) and dystocial mare (n=14), foal serum IgG was lower in foals born in dystocia than in foals in normal foaling (P<0.05). This study demonstrates reference values for colostral and foal serum IgG specific to heavy draft horses in Japan and suggests that dystocia may interfere with the acquisition of colostral immunity in neonatal foals.

Single-Layer Colloid Centrifugation as a Method to Process Urine-Contaminated Stallion Semen After Freezing-Thawing.

Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine contamination. Semen was extended 1:1, cushion-centrifuged, and resuspended at 200 million sperm/mL in BotuCrio. Resuspended semen was loaded in 0.5 mL straws and cryopreserved in liquid nitrogen. Samples were thawed (37°C for 30 seconds) and processed by SLC (400 g/30 minutes). Percentages of total motility (TM) and progressive motility (PM) were assessed with computer-assisted semen analyzer. Sperm viability (%VIAB) and yield were assessed with a NucleoCounter before and after gradient centrifugation. Data were analyzed with two-way ANOVA and Tukey's test. The motility parameters TM before SLC (control: 35 ± 2; low: 33 ± 0.7; high: 22 ± 1.8) after SLC (control: 51 ± 3.6; low: 42 ± 2.2; high: 25 ± 2.8) and PM before SLC (control: 24 ± 1.8; low: 21 ± 1.14; high: 12 ± 1.5) and after SLC (control: 40.3 ± 3.2; low: 31 ± 3.9; high: 14 ± 2) significantly decreased with increasing urine contamination. Urine contamination marginally reduced (P < .05) sperm viability after cryopreservation before SLC (control: 45 ± 0.7; low: 27 ± 0.2; high: 27 ± 0.3) and after SLC (control: 54 ± 0.5; low: 49 ± 0.7; high: 38 ± 0.6). Recovery rates of sperm after centrifugation were not significantly different between groups. In conclusion, urine contamination affects sperm motility parameters in a dose-dependent manner. Post-thaw SLC selected sperm with higher motility and viability in control and low groups but only selected sperm with higher viability in the high group.

Impacts of oocyte/zygote timing for in vitro fertilization and gene editing in the dog.

Previously, we reported the first live births of dogs using in vitro fertilization (IVF), embryo cryopreservation, and transfer. These techniques have potential applications in the conservation of endangered canids, and development of gene editing/repair technologies that could improve animal welfare by restoring normal gene function and removing predisposition to disease. Here, we used IVF as a springboard for initial attempts at genetic modification through gene editing/repair using the Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated endonuclease (Cas9) system. We showed previously that timing is critical for successful IVF in that the canine oocyte must be exposed to the oviductal environment beyond simply reaching metaphase II. Others have shown that timing of injection of CRISPR-Cas9 constructs is critical in gene editing, influencing the extent of genetic mosaicism. Therefore, we investigated whether timing of injection of the gene editing/repair constructs might influence the success of embryo production and gene editing in the dog. We achieved similar IVF success to our prior report in generating 2-cell control embryos, and found equally reduced embryo production whether injection was performed in oocytes prior to fertilization, or in presumptive single-cell zygotes already exposed to sperm. We had no success at generating offspring with precise single-nucleotide changes in KRT71 via homology-directed repair (HDR), but did identify mutation of FGF5 using non-homologous end joining (NHEJ). These findings underscore the difficulties inherent to gene repair, but represent important progress on reproducibility of canine IVF, improved techniques of oocyte/embryo handling, and impact of timing of injections on embryo development.