Success rates and factors influencing pregnancy outcome after 464 transvaginal ultrasound-guided twin reductions in the mare.

BACKGROUND: Transvaginal ultrasound-guided aspiration (TUA) is used for post-fixation twin reduction in mares. However, there is limited information regarding factors that influence pregnancy outcome after TUA. OBJECTIVES: To evaluate the effect of day of gestation on which TUA is performed, aspiration volume, puncture of the conceptus, medication administered before and after TUA, embryo location, mare age and parity and operator experience on pregnancy and foaling rates after TUA. STUDY DESIGN: Retrospective case series. METHODS: Data were collected from case records of 464 TUAs performed by 14 operators in 422 mares diagnosed pregnant with dizygotic twins in two different facilities between 2010 and 2019. Pregnancy status was determined by ultrasonography at 5-7 days and 3-4 weeks after the TUA was performed. Subsequent pregnancy and foaling results were obtained by follow-up communication. The effects of mare, gestation- and TUA-related variables on pregnancy and foaling rates were analysed by the chi-square-test for homogeneity and Fisher's exact test and logistic regression. RESULTS: TUA was performed between 21 and 82 days of gestation in unilaterally (267/359 [74.4%]) and bilaterally fixed (92/359 [25.6%]) twin pregnancies. A singleton pregnancy (218/381 [57.2%]), persistent twin pregnancy (60/381 [15.8%]), or the loss of both conceptuses (103/381 [27%]) was confirmed 5-7 days after TUA was performed. At 3-4 weeks post TUA 50.3% (163/324) of mares were diagnosed with a single viable pregnancy and 40.1% (127/317) went on to deliver a live single foal. TUA performed early in gestation (D 25-35) resulted in the birth of a live singleton foal in 49.3% (74/150) of mares. MAIN LIMITATIONS: Missing retrospective data despite extensive follow-up. CONCLUSION: This is the first large scale study to demonstrate that acceptable pregnancy and foaling rates can be achieved in mares diagnosed with twins when TUA is performed early in gestation (<40 days).

Bovine oviductal organoids: a multi-omics approach to capture the cellular and extracellular molecular response of the oviduct to heat stress.

BACKGROUND: The mammalian oviduct is a complex, fibromuscular organ known for its role in orchestrating a series of timely and dynamic changes to suitably support early embryogenesis. Climate change-induced heat stress (HS) is one of the largest single stressors compromising reproductive function in humans and farm animals via systemic changes in the redox status of the maternal environment, adversely affecting fertilization and early embryonic development. Oviductal organoids represent a unique 3-dimensional, biomimetic model to study the physiology of the oviduct and its subsequent impact on embryo development under various environmental conditions. RESULTS: Our study is the first to demonstrate an innovative approach to understanding the cascade of molecular changes sustained by bovine oviductal organoids under HS and the subsequent maternal signals harnessed within their secreted extracellular vesicles (EVs). Transcriptomic analysis of oviductal organoids exposed to HS revealed 2,570 differentially expressed genes (1,222 up- and 1,348 downregulated), while EV-coupled miRNome analysis disclosed 18 miRNAs with significant differential expression (12 up- and 6 downregulated) in EVs from thermally stressed organoids compared to EVs released from organoids cultured under thermoneutral conditions. Genes activated in oviductal organoids in response to thermal stress, include: COX1, ACTB, CST6, TPT1, and HSPB1, while miR-1246, miR-148a, miR21-5p, miR-451, and miR-92a represent the top highly abundant EV-coupled miRNAs released in response to HS. Pathway analysis of genes enriched in organoids exposed to thermal stress showed the enrichment of endocrine resistance, cellular senescence, and notch signaling pathways. Similarly, EV-coupled miRNAs released from thermally stressed organoids showed their potential regulation of genes involved in cellular senescence, p53 signaling, and TGF-beta signaling pathways. CONCLUSIONS: In conclusion, the cellular and extracellular response of bovine oviductal organoids to in vitro HS conditions reveal the prospective impact of environmental HS on the physiology of the oviduct and the probable subsequent impacts on oocyte fertilization and early embryo development. Future studies elucidating the potential impact of HS-associated EVs from oviductal organoids on oocyte fertilization and preimplantation embryo development, would justify the use of an organoid model to optimally understand the oviduct-embryo communication under suboptimal environments.

Extracellular vesicles in the treatment and prevention of osteoarthritis: can horses help us translate this therapy to humans?

Osteoarthritis (OA) is a common joint disease affecting humans and horses, resulting in significant morbidity, financial expense, and loss of athletic use. While the pathogenesis is incompletely understood, inflammation is considered crucial in the development and progression of the disease. Mesenchymal stromal cells (MSCs) have received increasing scientific attention for their anti-inflammatory, immunomodulatory, and pro-regenerative effects. However, there are concerns about their ability to become a commercially available therapeutic. Extracellular vesicles (EVs) are now recognized to play a crucial role in the therapeutic efficacy observed with MSCs and offer a potentially novel cell-free therapeutic that may negate many of the concerns with MSCs. There is evidence that EVs have profound anti-inflammatory, immunomodulatory, and pro-regenerative effects equal to or greater than the MSCs they are derived from in the treatment of OA. Most of these studies are in small animal models, limiting the translation of these results to humans. However, highly translational animal models are crucial for further understanding the efficacy of potential therapeutics and for close comparisons with humans. For this reason, the horse, which experiences the same gravitational impacts on joints similar to people, is a highly relevant large animal species for testing. The equine species has well-designed and validated OA models, and additionally, therapies can be further tested in naturally occurring OA to validate preclinical model testing. Therefore, the horse is a highly suitable model to increase our knowledge of the therapeutic potential of EVs.

Anti-Müllerian hormone and inhibin-B concentrations vary cyclically in nonovulating queens within reference ranges established for determining gonadal status in cats.

OBJECTIVE: To define cyclic changes in anti-Müllerian hormone (AMH), inhibin-B, and progesterone concentrations and establish statistically valid, population-based clinical reference ranges in queens. ANIMALS: Cyclic queens (fertile, n = 6; infertile, 6) from an institutional breeding colony were blood sampled longitudinally, each for over 2 months, between November 2021 and February 2022, and residual serum samples from intact (n = 205) and ovariohysterectomized (49) queens from clinical submissions were used to establish reference ranges for intact and spayed females. METHODS: AMH and inhibin-B were measured using commercially available ELISAs, progesterone was measured using an in-house ELISA, and 90% CIs were calculated from these data. RESULTS: AMH and inhibin-B fluctuated in a highly correlated, cyclic pattern in 3 queens that did not ovulate immediately, whereas AMH declined as progesterone increased, indicative of ovulation, which occurred spontaneously early in the sampling period in 3 others; statistically valid reference ranges were established in intact and ovariohysterectomized females. CLINICAL RELEVANCE: Cyclic changes in hormone profiles were defined, providing relevant context for interpreting results in cases seeking to determine gonadal status (presence or absence of gonadal tissue) on the basis of established, population-based reference ranges reported here for cats for the first time.

Production of Mare Chorionic Girdle Organoids That Secrete Equine Chorionic Gonadotropin.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

Corticotropin releasing hormone is present in the feline placenta and maternal serum.

Background: In women, placental corticotropin releasing hormone (CRH) can be detected in maternal blood throughout pregnancy and is important in the regulation of the timing of parturition. However, its role in other mammalian species is unclear. In fact, very little is known about the presence and localization of CRH in placentas other than human. In this study we report for the first time the presence of CRH in feline placenta and maternal serum. Methods: Presence of CRH mRNA and protein was assessed using RT-PCR and Western blot, respectively, in at term domestic cat placentas opportunistically obtained at a local animal shelter and spay clinic. In addition, CRH localization within the placenta was demonstrated via immunohistochemistry. Finally, presence of CRH in maternal blood from early (¾21 days) and mid (25-35 days) stages of pregnancy was investigated by ELISA. Results: CRH mRNA and protein were detected in feline placentas, and localized to larger decidual cells and fetal trophoblast cells, including the binucleate cells. CRH was detectable in maternal blood collected from early-stage pregnancies, and amounts significantly increased in mid-gestation samples. Conclusion: This is the first report on the presence and localization of CRH in the feline placenta, and its increase in maternal serum during the first half of pregnancy. These data lay the foundation for future studies to determine if CRH can be used as potential novel marker for early pregnancy diagnosis, determination, and monitoring in felids, and could greatly increase efficiency and success in zoo breeding programs utilizing artificial reproductive technologies for endangered feline species.

Generation and cryopreservation of feline oviductal organoids.

Next-generation in vitro culture model systems are needed to study the reproductive pathologies that affect domestic animals. These 3D culture models more closely mimic normal physiological function to allow a greater understanding of reproductive pathology and to trial therapeutics without the welfare concerns and the increased time and cost associated with live animal research. Recent advances with in vitro cell culture systems utilizing human and laboratory animal tissues have been reported, but implementation of these technologies in veterinary species has been slower. Organoids are a physiologically representative 3D cell culture system that can be maintained long-term. By combining organoid culture with cryopreservation, a long-term, experimental model can be available for year-round application, thus bypassing seasonality and reproductive tract availability restrictions. Here we report the generation and cryopreservation of feline oviductal organoids for the first time. Optimal culture medium for the generation of feline oviductal organoids was established, and organoids were successfully cryopreserved using three different freezing media with organoids from each treatment demonstrating comparable viability, growth rate, and protein expression after thawing and culture. Feline oviductal organoids may facilitate an in vivo-like environment that, in conjunction with co-culture for in vitro maturation and in vitro fertilization, may positively influence in vitro gamete and embryo development, embryo quality, and pregnancy rates after embryo transfer in domestic and nondomestic felids. Furthermore, readily available cryopreserved feline oviductal organoids will facilitate this co-culture, which is of particular importance to endangered felid breeding programs where tissue and gamete samples are often opportunistically obtained with little or no notice.

The Correlation of Endogenous Progesterone Concentration in Diestrus on Early Pregnancy Rate in Thoroughbred Mares.

The aim of this project was to test the hypothesis that progesterone concentration 5 days after ovulation did not differ between pregnant and nonpregnant Thoroughbred mares on stud farms located in the Waikato region of New Zealand. A prospective cohort study was performed involving five stud farms in the Waikato region of New Zealand during the 2018 breeding season. A total of 275 mares were enrolled in the study. Mares were served by 34 individual stallions. Blood samples were taken from each mare 5 days after ovulation (D0) and measured for progesterone concentration. Early pregnancy was confirmed at D14 by transrectal palpation and ultrasonography of the mares reproductive tract. Progesterone concentration at Day 5 post-ovulation was higher in mares determined to pregnant at Day 14 of gestation than in mares determined to be non-pregnant at Day 14 (6.4 ± 3.0 ng/ml vs. 5.5 ± 3.3 ng/ml respectively; P = .02). A negative association between increasing mare age and pregnancy rate was found but mare age had no effect on progesterone concentration at D5. In this study we found that although higher serum progesterone concentration at Day 5 post ovulation was associated with a higher pregnancy rate at Day 14, no predictive or definitive minimum required progesterone concentration could be identified. Additional studies are required to determine if a synthetic progestogens can serve to supplant natural progesterone to increase pregnancy rate in naturally bred mares.

Production of extracellular vesicles from equine embryo-derived mesenchymal stromal cells.

In brief: Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promise as off-the-shelf therapeutics; however, producing them in sufficient quantities can be challenging. In this study, MSCs were isolated from preimplantation equine embryos and used to produce EVs in two commercially available bioreactor designs. Abstract: Mesenchymal stromal cells (MSC) have recently been explored for their potential use as therapeutics in human and veterinary medicine applications, such as the treatment of endometrial inflammation and infertility. Allogeneic MSC-derived extracellular vesicles (EVs) may also provide therapeutic benefits with advantage of being an 'off-the-shelf' solution, provided they can be produced in large enough quantities, without contamination from bovine EVs contained in fetal bovine serum that is a common component of cell culture media. Toward this aim, we demonstrated the successful isolation and characterization of equine MSCs from preimplantation embryos. We also demonstrate that many of these lines can be propagated long-term in culture while retaining their differentiation potential and conducted a head-to-head comparison of two bioreactor systems for scalable EV production including in serum-free conditions. Based on our findings, the CELLine AD 1000 flasks enabled higher cell density cultures and significantly more EV production than the FiberCell system or conventional culture flasks. These findings will enable future isolation of equine MSCs and the scalable culture of their EVs for a wide range of applications in this rapidly growing field.

Effect of Acetylsalicylic Acid on Uterine Blood Flow, Gestation Length, Foal Birth Weight and Placental Weight in Pregnant Thoroughbred Mares - A Clinical Pilot Study.

The aim of this double-blinded placebo-controlled study was to investigate the effect of acetylsalicylic acid (ASA) on uterine blood flow, gestation length, placental and foal weights in pregnant mares. Sixteen Thoroughbred mares of different age (13.3 ± 4.1) and parity (7.4 ± 3.1) were randomly assigned to three treatment groups. Mares in group C (n = 4) served as controls and received 5,000 mg lactose orally once daily from D 120 (D 0 = day of ovulation) until parturition. Mares in group ASA1 (n = 7) received 5,000 mg ASA orally once daily from D 120 until parturition. Mares in group ASA2 (n = 5) received the same dose ASA as group ASA1 from D 120 to D 285, but twice daily from D 285 until parturition. Mares were examined by ultrasonography on D 14, 28, and 60, and in 21-days intervals from D 120 until parturition. The cross-sectional area, time average maximum velocity (TAMV), and pulsatility index were measured in both uterine arteries and the blood flow volume was calculated for each uterine artery and then summarized. All 16 mares carried a normal pregnancy and delivered live foals. In group ASA2 TAMV in the ipsilateral artery was significantly higher (P = .03) and these mares showed a tendency of increased total blood flow volume (P = .07) during late pregnancy (D 305-346). Results indicate that oral administration of 5,000 mg of ASA twice daily in pregnant mares causes a rise in uterine blood flow during late pregnancy.

Equine Oviductal Organoid Generation and Cryopreservation.

Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze-thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of their normal physiology, including hormonal regulation. These organoids may provide an environment that mimics the in vivo equine oviduct and facilitate improved in vitro embryo production in equids.

The Roles of Extracellular Vesicles and Organoid Models in Female Reproductive Physiology.

Culture model systems that can recapitulate the anatomy and physiology of reproductive organs, such as three-dimensional (3D) organoid culture systems, limit the cost and welfare concerns associated with a research animal colony and provide alternative approaches to study specific processes in humans and animals. These 3D models facilitate a greater understanding of the physiological role of individual cell types and their interactions than can be accomplished with traditional monolayer culture systems. Furthermore, 3D culture systems allow for the examination of specific cellular, molecular, or hormonal interactions, without confounding factors that occur with in vivo models, and provide a powerful approach to study physiological and pathological reproductive conditions. The goal of this paper is to review and compare organoid culture systems to other in vitro cell culture models, currently used to study female reproductive physiology, with an emphasis on the role of extracellular vesicle interactions. The critical role of extracellular vesicles for intercellular communication in physiological processes, including reproduction, has been well documented, and an overview of the roles of extracellular vesicles in organoid systems will be provided. Finally, we will propose future directions for understanding the role of extracellular vesicles in normal and pathological conditions of reproductive organs, utilizing 3D organoid culture systems.

Evaluation of growth, viability, and structural integrity of equine endometrial organoids following cryopreservation.

Reproductive diseases in mares are a significant cause of subfertility and profound economic loss in the equine industry. Utilizing a 3D in vitro cell culture system that recapitulates the in vivo physiology will reduce time, cost, and welfare concerns associated with in vivo reproductive research in mares. If this 3D model is combined with effective cryopreservation, reproductive research on mares can occur year-round, which is not currently possible in this seasonal species. Endometrial organoids, 3D in vitro cell clusters that exhibit in vivo uterine physiology, have been established in mice, women, and mares. Here we report the first comprehensive assessment of cryopreservation of endometrial organoids in the domestic mare. Organoid growth rate was not affected by the type of freezing media. However, growth rate varied among non-cryopreserved controls, organoids cryopreserved at passage 0 (P0), and organoids cryopreserved at passage 3 (P3). Additionally, there was no difference in organoid viability among freezing media or freezing timepoint (passages). Furthermore, fresh and frozen-thawed organoids displayed positive immunohistochemical staining for ZO-1, which is a marker for intercellular tight junctions, and for periodic acid-Schiff staining as marker for organoid function through mucin production. Results demonstrate that equine endometrial organoids can be cryopreserved with 10% dimethyl sulfoxide with minimal detrimental effects while maintaining intercellular tight junctions (ZO-1) and secretory function. Availability of cryopreserved endometrial organoids may permit expanded research on uterine pathologies that negatively affect mare fertility and improve efficiency, reduce cost, and minimize animal welfare concerns associated with in vivo research in the domestic mare.

Pyometra in the queen: To spay or not to spay?

PRACTICAL RELEVANCE: Pyometra is a commonly occurring uterine disease in cats that often leads to loss of breeding potential and, in some cases, can be life threatening. An increased incidence of cystic endometrial hyperplasia (CEH) and pyometra is seen with age. Most queens present with uterine lesions after 5-7 years of age (average 7.6 years, range 1-20 years). Clinical signs most commonly occur within 4 weeks of the onset of oestrus in queens that are either mated, spontaneously ovulate or are induced to ovulate (mechanical stimulation or hormone induction). The disease is most often observed in dioestrus. CLINICAL CHALLENGES: Queens with pyometra often go undiagnosed as there may be few or only very mild clinical signs and laboratory changes. For example, the classic sign of mucopurulent bloody vulvar discharge often goes unnoticed. Abdominal ultrasound is the best tool for diagnosis of pyometra and for monitoring response to therapy. PATIENT GROUP: Classically, middle-aged/older nulliparous intact queens present with pyometra. However, so-called 'stump pyometra' can occur if ovarian tissue is left behind during ovariectomy or ovariohysterectomy (ovarian remnant syndrome). Queens treated with exogenous steroid hormones such as high doses of megestrol acetate or medroxyprogesterone acetate for oestrus prevention can also develop CEH and pyometra. EVIDENCE BASE: There has been little published to date on CEH, endometritis and pyometra in the queen and most of the currently available information has been extrapolated from studies carried out in the bitch. The queen and the bitch have very different reproductive physiology; thus, further research and investigation into the precise aetiopathogenesis of these disease processes of the uterus in the queen is warranted. AUDIENCE: This review is aimed at clinicians working in small animal practice, especially those in countries where surgical sterilisation is not practised as commonly as in the United States, Canada or Australasia, and who will therefore see a greater proportion of intact queens.

Effects of age, parity, and pregnancy abnormalities on foal birth weight and uterine blood flow in the mare.

Color Doppler sonography has become routine for the evaluation of high-risk pregnancies in human medicine. Previous studies documenting uterine blood flow parameters in the pregnant mare have found a decrease in peripheral blood flow resistance in the first pregnancy weeks and an increase in uterine blood flow, especially in the last trimester of pregnancy. However, these studies involved only a small number of mares. No naturally occurring pregnancy abnormalities occurred that would allow blood flow changes to be retrospectively examined and analyzed. The objective of the present study was to monitor the diameter of the uterine artery, uterine blood flow, and the combined thickness of the uterus and placenta (CTUP) throughout gestation in a large number of pregnant mares of different age and parity. In the present study, 51 warmblood mares were examined by ultrasonography on Days 16 and 30, at monthly intervals until Day 300, and then every 10 days from Day 300 until parturition. After localization of the uterine artery ipsilateral and contralateral to the conceptus, the diameter of each artery, the uterine blood flow (pulsatility index [PI], blood flow volume [BFV], and the presence of early diastolic notch), and the CTUP were measured and correlated to placental and foal birth weight after delivery. Furthermore, the effect of age (3-7 years [n = 16], 8-11 years [n = 17], 12-16 years [n = 18]) and parity (0-2 foals [n = 22], 3-4 foals [n = 15], 5-8 foals [n = 14]) on these parameters were analyzed. The diameter of the uterine artery increased more than threefold in the ipsilateral artery (0.40 ± 0.07-1.33 ± 0.08 cm) and 2.7-fold in the contralateral artery (0.39 ± 0.07-1.07 ± 0.08 cm [P < 0.0001]). The early diastolic notch disappeared in the pulse waves in 98% of the ipsilateral arteries and 85.7% in the contralateral arteries on Day 150 when placentation is complete. Blood flow volume increased 50-fold in the ipsilateral artery during pregnancy and increased dramatically in the last trimester. The median foal weight was 52.6 kg. Mares with heavier foals (>52.6 kg) had a 1.38-fold higher BFV in the last 2 months (P < 0.05) compared with lighter foals. Pulsatility index decreased 2-fold until completion of placentation at around Day 150 and continued to decline until Day 240 where it then stayed constant and at a low level until delivery. Age predominantly influenced PI, whereas the diameter of the uterine arteries, which is correlated to BFV (r ipsilateral = 0.919, P < 0.0001 and r contralateral = 0.909, P < 0.000), was strongly affected by parity. Four mares spontaneously aborted (Days 200, 208, 213, and 246) and four mares spontaneously developed placentitis that was diagnosed by the presence of an increased CTUP and/or placental pathology after delivery. Although not statistically relevant, the aborting mares showed a slightly increased total BFV, but no differences in PI were seen compared with mares without abnormalities of pregnancy. Mares that developed placentitis had a late (Days 150-210) disappearance of the early diastolic notch and an increased PI in the first half of pregnancy. In conclusion, the study documented differences in uterine artery diameter and blood flow in a large number of pregnant mares. Furthermore, this is the first known report to document uteroplacental blood flow changes associated with naturally occurring placentitis and abortion in mares.

The effect of gamete co-incubation time during in vitro fertilization with frozen-thawed unsorted and sex-sorted ram spermatozoa on the development of in vitro matured adult and prepubertal ewe oocytes.

In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubated with unsorted or sex-sorted frozen-thawed spermatozoa for 2 to 3 h (short) or 18 to 20 h (long) to determine the effects of reducing the gamete co-incubation time during IVF on subsequent embryonic development in vitro. For oocytes derived from adult ewes, there were no differences in oocyte fertilization and cleavage at 24 h post insemination (hpi) between types of spermatozoa or co-incubation times (P > 0.05). By 48 hpi, oocyte cleavage was higher after a short (390/602, 64.8%) compared with a long (381/617, 61.7%) co-incubation (P < 0.05), and was not significantly different for unsorted (266/372, 71.5%) and sex-sorted (505/849, 59.9%) spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (150/266, 56.4%) and sex-sorted (295/505, 58.4%) spermatozoa, but was higher after a short (240/390, 61.5%) than long (205/381, 53.8%) co-incubation (P < 0.05). Oocyte development to the blastocyst stage was not different for unsorted (150/372; 40.3%) and sex-sorted (295/847; 34.8%) spermatozoa but was significantly increased by a short (240/602, 39.9%) compared with a long (205/617, 33.2%) co-incubation. Fertilization of oocytes from prepubertal ewes was similar for types of spermatozoa and for duration of co-incubation. Oocyte cleavage (48 hpi) was similar for a short (241/377, 63.9%) and long (226/349, 64.8%) co-incubation with unsorted spermatozoa, but was increased (P < 0.05) by a long co-incubation (286/500, 57.2% versus 163/517, 31.5%) with sex-sorted spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (230/467, 49.3%) and sex-sorted (186/449, 41.4%) spermatozoa, and a short (200/404, 49.5%) or long (216/512, 42.1%) co-incubation. However, oocyte development to the blastocyst stage was higher (P < 0.05) after IVF with unsorted (230/726, 37.1%) than sex-sorted (186/1017, 18.3%) spermatozoa. Reducing the duration of gamete co-incubation did not deleteriously affect the in vitro development of adult and prepubertal ewe derived oocytes after IVF with unsorted and sex-sorted spermatozoa. In general, sex-sorting had no substantial influence on fertilization and embryo development rates.

Flow cytometric sorting of non-human primate sperm nuclei.

Pre-determination of the sex of offspring has implications for management and conservation of captive wildlife species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort spermatozoa of non-human primate species for use with assisted reproductive technologies. The objectives of this study were to: (i) determine the difference in DNA content between X- and Y-bearing spermatozoa (ii) sort sperm nuclei into X- and Y-enriched samples; and (iii) assess the accuracy of sorting. Spermatozoa were collected from two common marmosets (Callithrix jacchus), seven hamadryas baboons (Papio hamadryas) and two common chimpanzees (Pan troglodytes). Human spermatozoa from one male were used as a control. Sperm nuclei were stained (Hoechst 33342), incubated and analyzed using a high-speed cell sorter. Flow cytometric reanalysis of sorted samples (sort reanalysis, 10,000 events/sample) and fluorescence in situ hybridization (FISH; 500 sperm nuclei/sample) were used to evaluate accuracy of sorting. Based on fluorescence intensity of X- and Y-bearing sperm nuclei, the difference in DNA content between X and Y populations was 4.09 +/- 0.03, 4.20 +/- 0.03, 3.30 +/- 0.01, and 2.97 +/- 0.05%, for marmoset, baboon, chimpanzee and human, respectively. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94 +/- 0.9 and 93 +/- 0.8%, 94 +/- 0.7 and 94 +/- 0.5%, 91 +/- 0.9 and 97 +/- 0.6%, 94 +/- 0.6 and 94 +/- 0.9%, for marmoset, baboon, chimpanzee and human, respectively). These data indicate the potential for high-purity sorting of spermatozoa from non-human primates.