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GPS collars have revolutionized the field of animal ecology, providing detailed information on animal movement and the habitats necessary for species survival. GPS collars also have the potential to cause adverse effects ranging from mild irritation to severe tissue damage, reduced fitness, and death. The impact of GPS collars on the behavior, stress, or activity, however, have rarely been tested on study species prior to release. The objective of our study was to provide a comprehensive assessment of the short-term effects of GPS collars fitted on scimitar-horned oryx (Oryx dammah), an extinct-in-the-wild antelope once widely distributed across Sahelian grasslands in North Africa. We conducted behavioral observations, assessed fecal glucocorticoid metabolites (FGM), and evaluated high-resolution data from tri-axial accelerometers. Using a series of datasets and methodologies, we illustrate clear but short-term effects to animals fitted with GPS collars from two separate manufacturers (Advanced Telemetry Systems-G2110E; Vectronic Aerospace-Vertex Plus). Behavioral observations highlighted a significant increase in the amount of headshaking from pre-treatment levels, returning below baseline levels during the post-treatment period (>3 days post-collaring). Similarly, FGM concentrations increased after GPS collars were fitted on animals but returned to pre-collaring levels within 5 days of collaring. Lastly, tri-axial accelerometers, collecting data at eight positions per second, indicated a > 480 percent increase in the amount of hourly headshaking immediately after collaring. This post-collaring increase in headshaking was estimated to decline in magnitude within 4 hours after GPS collar fitting. These effects constitute a handling and/or habituation response (model dependent), with animals showing short-term responses in activity, behavior, and stress that dissipated within several hours to several days of being fitted with GPS collars. Importantly, none of our analyses indicated any long-term effects that would have more pressing animal welfare concerns.

Plastic polymers can be combined with additives that modify physical properties and stability of the material. However, the biocompatibility of those additives is not well known. The objective of the study was to characterize the impact of zinc stearate-a common additive-through the development of a novel three-dimensional (3-D) in vitro platform with endometrial cells from domestic cats. Epithelial and stromal cells from adult uteri were isolated and cultured in medium supplemented with 3% Matrigel for two weeks in plastic tissue culture dishes that had been identified as polystyrene with and without zinc stearate by Raman, FTIR, and X-ray fluorescence spectroscopies. Three-dimensional cell structures that were obtained were measured and categorized by shape. Cell viability, proliferation, differentiation, organization, and apoptosis then were assessed by immuno-staining. Results indicated that zinc stearate did not affect 3-D endometrial cell structure morphology, viability, or cellular composition. This first study of a new in vitro platform will be useful for studies testing the influence of other additives, drugs, or exogenous hormones.

Ovarian tissue contains large pools of immature oocytes enclosed in primordial follicles, making it an attractive target for fertility preservation in female cancer patients, livestock and wild species. Compared to cryopreservation, desiccation and long-term storage of samples at supra-zero temperatures (using strategies inspired from small organisms to resist extreme environments) would be more cost-effective and convenient. The objective of the study was to characterize the influence of microwave-assisted dehydration on structural and functional properties of living ovarian tissues. While this method allows preservation of single cells (cat oocytes and sperm cells so far) using trehalose as the xeroprotectant, it has not been developed for multicellular tissues yet. Ovarian cortex biopsies were reversibly permeabilized, exposed to various concentrations of trehalose, and dried for different times using a commercial microwave under thermal control. Effective dehydration of samples along with proper trehalose retention were reached within 30 min of microwave drying. Importantly, the process did not affect morphology and DNA integrity of follicles or stromal cells. Moreover, transcriptional activity and survival of follicles were partially maintained following 10 min of drying, which already was compatible with storage at non-cryogenic temperatures. Present data provide critical foundation to develop dry-preservation techniques for long-term storage of living multicellular tissues.

The present study evaluated the effect of supplementing in vitro culture medium with J. insularis compared to FSH on isolated secondary follicles and in vitro maturation of oocytes from those follicles. Secondary follicles were isolated from sheep ovaries and individually cultured for 18 days in α-MEM+ (Control), α-MEM+ supplemented with 100 ng/mL recombinant bovine follicle stimulating hormone (FSH) or with 0.3, 1.25, or 2.5 mg/mL of J. insularis extract (JI0.3, JI1.25, and JI2.5, respectively). Culture medium collected every 2 days was used to measure ROS levels. At the end of the culture period, cumulus oocytes complex (COCs) were collected and matured in vitro. Follicular walls were used for mRNA quantitation. JI0.3 led to a higher (P < 0.05) percentages of intact follicles than other groups after 18 days of culture. While follicular diameter remained unchanged from Day 6 onwards with JI0.3 and FSH, percentages of antral cavity formation were higher (P < 0.05) with JI0.3 at Day 6 than in all other treatments. No differences were observed between controls and treatment groups regarding ROS levels and mRNA expression of genes. Viability of resulting oocytes was higher (P < 0.05) in JI0.3 compared to FSH. Interestingly, in control experiment, supplementation of maturation medium with JI0.3 led to higher (P < 0.05) percentages of metaphase II compared to controls. Although more validations will be needed, it seems that this natural extract could be used as a cheap and easily available alternative to commercial FSH.

Retinoic acid (RA) facilitates tissue morphogenesis by regulating matrix matalloproteinase (MMPs) expression. Our objective was to examine the influence of RA on in vitro development of follicles enclosed within domestic cat ovarian tissues. Ovarian cortices from 9 prepubertal and 13 adult cats were incubated for 7 d in medium containing 0 (control), 1 or 5 μM RA and then analyzed for viability. Cortices from additional three animals of each age group were cultured in the same condition and follicle morphology, stage and size were histologically evaluated. In a separate study, cortices from 14 donors (7 prepubertal; 7 adult cats) were incubated in 0 or 5 μM RA for 7 d and assessed for (1) MMP1, 2, 3, 7, 9 and TIMP1 expression by qPCR and (2) protein expression of MMP9 by immunohistochemistry. Donor age did not influence follicle response to RA. Collective data from both age groups revealed that percentages of primordial follicles in 5 μM RA treatment were lower (P < 0.05; 40.5 ± 4.5%) than in fresh cortices (66.7 ± 5.3%) or controls (60.1 ± 4.0%) with 1 μM-RA treatment producing intermediate (56.3 ± 4.0%) results. Proportion of primary follicles in 5 μM RA (21.7 ± 3.3%) was higher than in fresh cortices (4.9 ± 2.9%) and controls (9.0 ± 2.8%) with 1 μM-RA treatment producing an intermediate value (13.8 ± 2.0%). Furthermore, proportion of secondary follicles increased after 7 d in the presence of 5 μM RA (9.5 ± 2.7%) compared to other groups (fresh, 1.9 ± 0.8%; control, 2.6 ± 1.1%; 1 μM RA, 2.5 ± 0.2%). MMP9 transcript and protein were upregulated, whereas MMP7 mRNA was suppressed by 5 μM-RA treatment compared to fresh counterparts. RA did not impact MMP1, 2, 3, 13 or TIMP1 expression. In summary, RA activated cat primordial follicle growth likely via a mechanism related to upregulation of MMP9 and down-regulation of MMP7 transcripts.

Understanding critical roles of warming and reanimation is critical to improve the survival of vitrified testicular tissue in domestic cats. The objective was to study structural and functional properties of testicular tissues from prepubertal domestic cats after standard vitrification followed by two warming protocols (directly at 37°C or with a 5-second pre-exposure to 50°C) and three reanimation time points (immediately, 24 h and 5 days post-warming). In Experiment 1, tissues were evaluated for histo-morphology and mitochondrial activity immediately or 24 h after warming protocols. In Experiment 2, cell viability, DNA fragmentation, and germ cell composition were assessed immediately, 24 h, or 5 days after optimal warming. Preservation of seminiferous tubule structure was better using warming at 50°C for five seconds, and survival of somatic as well as germinal cells was higher compared to direct warming at 37°C for one minute. Short term in vitro culture (for reanimation) also proved that cellular composition and functionality were better preserved when warmed for a short time at 50°C. Collective data showed that short warming at 50°C led to better quality of seminiferous tubule structure and cell composition after vitrification and short-term culture. In addition, data suggest clear directions to further understand and optimize testicular tissue survival after fertility preservation procedures.

Approximately 80% of cheetahs living in typical zoological collections never reproduce. In more than 60% of breedings, the female is confirmed to ovulate, but parturition fails to occur. It is unknown if these non-pregnant intervals of elevated progesterone (deemed luteal phases) are conception failures or a pregnancy terminating in embryonic/fetal loss. There have been recent advances in metabolic profiling and proteome analyses in many species with mass spectrometry used to identify 'biomarkers' and mechanisms indicative of specific physiological states (including pregnancy). Here, we hypothesized that protein expression in voided cheetah feces varied depending on pregnancy status. We: 1) identified the expansive protein profile present in fecal material of females; and 2) isolated proteins that may be candidates playing a role in early pregnancy establishment and diagnosis. Five hundred and seventy unique proteins were discovered among samples from pregnant (n = 8), non-pregnant, luteal phase (n = 5), and non-ovulatory control (n = 5) cheetahs. Four protein candidates were isolated that were significantly up-regulated and two were down-regulated in samples from pregnant compared to non-pregnant or control counterparts. One up-regulated candidate, immunoglobulin J chain (IGJ; an important component of the secretory immune system) was detected using a commercially available antibody via immunoblotting. Findings revealed that increased IGJ abundance could be used to detect pregnancy successfully in >80% of 23 assessed females within 4 weeks after mating. The discovery of a novel fecal pregnancy marker improves the ability to determine reproductive, especially gestational, status in cheetahs managed in an ex situ insurance and source population.

GPS collars have revolutionized the field of animal ecology, providing detailed information on animal movement and the habitats necessary for species survival. GPS collars also have the potential to cause adverse effects ranging from mild irritation to severe tissue damage, reduced fitness, and death. The impact of GPS collars on the behavior, stress, or activity, however, have rarely been tested on study species prior to release. The objective of our study was to provide a comprehensive assessment of the short-term effects of GPS collars fitted on scimitar-horned oryx (Oryx dammah), an extinct-in-the-wild antelope once widely distributed across Sahelian grasslands in North Africa. We conducted behavioral observations, assessed fecal glucocorticoid metabolites (FGM), and evaluated high-resolution data from tri-axial accelerometers. Using a series of datasets and methodologies, we illustrate clear but short-term effects to animals fitted with GPS collars from two separate manufacturers (Advanced Telemetry Systems-G2110E; Vectronic Aerospace-Vertex Plus). Behavioral observations highlighted a significant increase in the amount of headshaking from pre-treatment levels, returning below baseline levels during the post-treatment period (>3 days post-collaring). Similarly, FGM concentrations increased after GPS collars were fitted on animals but returned to pre-collaring levels within 5 days of collaring. Lastly, tri-axial accelerometers, collecting data at eight positions per second, indicated a > 480 percent increase in the amount of hourly headshaking immediately after collaring. This post-collaring increase in headshaking was estimated to decline in magnitude within 4 hours after GPS collar fitting. These effects constitute a handling and/or habituation response (model dependent), with animals showing short-term responses in activity, behavior, and stress that dissipated within several hours to several days of being fitted with GPS collars. Importantly, none of our analyses indicated any long-term effects that would have more pressing animal welfare concerns., Competing Interests: The authors have declared that no competing interests exist.

Ovarian tissue contains large pools of immature oocytes enclosed in primordial follicles, making it an attractive target for fertility preservation in female cancer patients, livestock and wild species. Compared to cryopreservation, desiccation and long-term storage of samples at supra-zero temperatures (using strategies inspired from small organisms to resist extreme environments) would be more cost-effective and convenient. The objective of the study was to characterize the influence of microwave-assisted dehydration on structural and functional properties of living ovarian tissues. While this method allows preservation of single cells (cat oocytes and sperm cells so far) using trehalose as the xeroprotectant, it has not been developed for multicellular tissues yet. Ovarian cortex biopsies were reversibly permeabilized, exposed to various concentrations of trehalose, and dried for different times using a commercial microwave under thermal control. Effective dehydration of samples along with proper trehalose retention were reached within 30 min of microwave drying. Importantly, the process did not affect morphology and DNA integrity of follicles or stromal cells. Moreover, transcriptional activity and survival of follicles were partially maintained following 10 min of drying, which already was compatible with storage at non-cryogenic temperatures. Present data provide critical foundation to develop dry-preservation techniques for long-term storage of living multicellular tissues., Competing Interests: The authors have declared that no competing interests exist.

Plastic polymers can be combined with additives that modify physical properties and stability of the material. However, the biocompatibility of those additives is not well known. The objective of the study was to characterize the impact of zinc stearate-a common additive-through the development of a novel three-dimensional (3-D) in vitro platform with endometrial cells from domestic cats. Epithelial and stromal cells from adult uteri were isolated and cultured in medium supplemented with 3% Matrigel for two weeks in plastic tissue culture dishes that had been identified as polystyrene with and without zinc stearate by Raman, FTIR, and X-ray fluorescence spectroscopies. Three-dimensional cell structures that were obtained were measured and categorized by shape. Cell viability, proliferation, differentiation, organization, and apoptosis then were assessed by immuno-staining. Results indicated that zinc stearate did not affect 3-D endometrial cell structure morphology, viability, or cellular composition. This first study of a new in vitro platform will be useful for studies testing the influence of other additives, drugs, or exogenous hormones., Competing Interests: The authors have declared that no competing interests exist.

The present study evaluated the effect of supplementing in vitro culture medium with J. insularis compared to FSH on isolated secondary follicles and in vitro maturation of oocytes from those follicles. Secondary follicles were isolated from sheep ovaries and individually cultured for 18 days in α-MEM+ (Control), α-MEM+ supplemented with 100 ng/mL recombinant bovine follicle stimulating hormone (FSH) or with 0.3, 1.25, or 2.5 mg/mL of J. insularis extract (JI0.3, JI1.25, and JI2.5, respectively). Culture medium collected every 2 days was used to measure ROS levels. At the end of the culture period, cumulus oocytes complex (COCs) were collected and matured in vitro. Follicular walls were used for mRNA quantitation. JI0.3 led to a higher (P < 0.05) percentages of intact follicles than other groups after 18 days of culture. While follicular diameter remained unchanged from Day 6 onwards with JI0.3 and FSH, percentages of antral cavity formation were higher (P < 0.05) with JI0.3 at Day 6 than in all other treatments. No differences were observed between controls and treatment groups regarding ROS levels and mRNA expression of genes. Viability of resulting oocytes was higher (P < 0.05) in JI0.3 compared to FSH. Interestingly, in control experiment, supplementation of maturation medium with JI0.3 led to higher (P < 0.05) percentages of metaphase II compared to controls. Although more validations will be needed, it seems that this natural extract could be used as a cheap and easily available alternative to commercial FSH., Competing Interests: The authors have declared that no competing interests exist.

Understanding critical roles of warming and reanimation is critical to improve the survival of vitrified testicular tissue in domestic cats. The objective was to study structural and functional properties of testicular tissues from prepubertal domestic cats after standard vitrification followed by two warming protocols (directly at 37°C or with a 5-second pre-exposure to 50°C) and three reanimation time points (immediately, 24 h and 5 days post-warming). In Experiment 1, tissues were evaluated for histo-morphology and mitochondrial activity immediately or 24 h after warming protocols. In Experiment 2, cell viability, DNA fragmentation, and germ cell composition were assessed immediately, 24 h, or 5 days after optimal warming. Preservation of seminiferous tubule structure was better using warming at 50°C for five seconds, and survival of somatic as well as germinal cells was higher compared to direct warming at 37°C for one minute. Short term in vitro culture (for reanimation) also proved that cellular composition and functionality were better preserved when warmed for a short time at 50°C. Collective data showed that short warming at 50°C led to better quality of seminiferous tubule structure and cell composition after vitrification and short-term culture. In addition, data suggest clear directions to further understand and optimize testicular tissue survival after fertility preservation procedures., Competing Interests: The authors have declared that no competing interests exist.

Retinoic acid (RA) facilitates tissue morphogenesis by regulating matrix matalloproteinase (MMPs) expression. Our objective was to examine the influence of RA on in vitro development of follicles enclosed within domestic cat ovarian tissues. Ovarian cortices from 9 prepubertal and 13 adult cats were incubated for 7 d in medium containing 0 (control), 1 or 5 μM RA and then analyzed for viability. Cortices from additional three animals of each age group were cultured in the same condition and follicle morphology, stage and size were histologically evaluated. In a separate study, cortices from 14 donors (7 prepubertal; 7 adult cats) were incubated in 0 or 5 μM RA for 7 d and assessed for (1) MMP1, 2, 3, 7, 9 and TIMP1 expression by qPCR and (2) protein expression of MMP9 by immunohistochemistry. Donor age did not influence follicle response to RA. Collective data from both age groups revealed that percentages of primordial follicles in 5 μM RA treatment were lower (P < 0.05; 40.5 ± 4.5%) than in fresh cortices (66.7 ± 5.3%) or controls (60.1 ± 4.0%) with 1 μM-RA treatment producing intermediate (56.3 ± 4.0%) results. Proportion of primary follicles in 5 μM RA (21.7 ± 3.3%) was higher than in fresh cortices (4.9 ± 2.9%) and controls (9.0 ± 2.8%) with 1 μM-RA treatment producing an intermediate value (13.8 ± 2.0%). Furthermore, proportion of secondary follicles increased after 7 d in the presence of 5 μM RA (9.5 ± 2.7%) compared to other groups (fresh, 1.9 ± 0.8%; control, 2.6 ± 1.1%; 1 μM RA, 2.5 ± 0.2%). MMP9 transcript and protein were upregulated, whereas MMP7 mRNA was suppressed by 5 μM-RA treatment compared to fresh counterparts. RA did not impact MMP1, 2, 3, 13 or TIMP1 expression. In summary, RA activated cat primordial follicle growth likely via a mechanism related to upregulation of MMP9 and down-regulation of MMP7 transcripts., Competing Interests: The authors have declared that no competing interests exist.

Approximately 80% of cheetahs living in typical zoological collections never reproduce. In more than 60% of breedings, the female is confirmed to ovulate, but parturition fails to occur. It is unknown if these non-pregnant intervals of elevated progesterone (deemed luteal phases) are conception failures or a pregnancy terminating in embryonic/fetal loss. There have been recent advances in metabolic profiling and proteome analyses in many species with mass spectrometry used to identify 'biomarkers' and mechanisms indicative of specific physiological states (including pregnancy). Here, we hypothesized that protein expression in voided cheetah feces varied depending on pregnancy status. We: 1) identified the expansive protein profile present in fecal material of females; and 2) isolated proteins that may be candidates playing a role in early pregnancy establishment and diagnosis. Five hundred and seventy unique proteins were discovered among samples from pregnant (n = 8), non-pregnant, luteal phase (n = 5), and non-ovulatory control (n = 5) cheetahs. Four protein candidates were isolated that were significantly up-regulated and two were down-regulated in samples from pregnant compared to non-pregnant or control counterparts. One up-regulated candidate, immunoglobulin J chain (IGJ; an important component of the secretory immune system) was detected using a commercially available antibody via immunoblotting. Findings revealed that increased IGJ abundance could be used to detect pregnancy successfully in >80% of 23 assessed females within 4 weeks after mating. The discovery of a novel fecal pregnancy marker improves the ability to determine reproductive, especially gestational, status in cheetahs managed in an ex situ insurance and source population.