RFRP3 influences basal lamina degradation, cellular death, and progesterone secretion in cultured preantral ovarian follicles from the domestic cat.

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies

UNIFORM SPERM MORPHOLOGY IN THE LEK-BREEDING WIRE-TAILED MANAKIN (PIPRA FILICAUDA)

Abstract ∙ When females copulate with multiple males, selection on spermatozoa can reduce variation in sperm morphology. We describe sperm morphology for a polygynous lek-breeding suboscine, the Wire-tailed Manakin (Pipra filicauda). Total sperm length averaged 41.5 ± 0.7 μm and the among-individual coefficient of variation in total sperm length was 1.8%. Variation was considerably lower than in the other manakin species with known sperm morphology, the Lance-tailed Manakin (Chiroxiphia lanceolata), despite similar promiscuity levels. This result highlights the need for further work on spermatozoa in lek-breeding species. Resumen ∙ Morfología uniforme en el esperma del Saltarín Uirapuru (Pipra filicauda), una especie con sistema de apareamiento de lek Cuando las hembras copulan con más de un macho, selección actuando al nivel del espermatozoide puede reducir la variación en la morfología del esperma. Aquí describimos la morfología del esperma para una especie poligínica de suboscín con sistema de apareamiento de lek, el Saltarín Uirapuru (Pipra filicauda). Los espermatozoides tuvieron una longitud total promedio de 41.5 ± 0.7 μm, y el coeficiente de variación para la longitud total fue de 1.8%. El nivel de variación fue menor que en la otra especie de saltarín estudiada al respecto, el Saltarín Lanceolado (Chiroxiphia lanceolata), aunque ambas especies tienen casi el mismo nivel de promiscuidad. Estos resultados sugieren la necesidad de más estudios sobre especies de aves con este sistema de apareamiento

In vitro exposure to epididymal extracellular vesicles from normospermic domestic cats improves developmental potential of sperm from teratospermic cats

We have previously reported a difference in the composition of epididymal extracellular vesicles (EVs) between normospermic and teratospermic domestic cats. The objective of the present study was to investigate whether the fertilizing ability or developmental potential of sperm from teratospermic cats could be improved after incubation with EVs isolated from normospermic cats. For each of 11 experimental replicates, pools of EVs were collected from the whole epididymides of 5 normospermic cats (normospermic EVs). Spermatozoa were also collected from the cauda epididymides of 2 teratospermic cats, pooled, and half was co-incubated with normospermic EVs for 1 h and 15 min prior to using the sperm for in vitro fertilization (IVF). The other half of the sperm was kept for 1 h and 15 min in the absence of EVs as a control group. We found no difference (p > 0.05) in sperm fertilizing ability, based on the percentage of cleaved embryos, after incubation with EVs (67.0%) and without EVs (60.6%). However, the developmental potential of teratospermic sperm, based on the proportion of embryos that reached the 8-cell stage or further, was better (p < 0.05) after co-incubation with EVs (58.4%) compared to the control group without EVs (47.2%). Additionally, the proportion of embryos that reached the blastocyst stage was better (p < 0.05) after co-incubation with EVs (30.7%) compared to the control group without EVs (19.9%). These findings can be used to improve the outcome of IVF with teratospermic males in domestic or wild felid species

Advancing stem cell technologies for conservation of wildlife biodiversity

Wildlife biodiversity is essential for healthy, resilient and sustainable ecosystems. For biologists, this diversity also represents a treasure trove of genetic, molecular and developmental mechanisms that deepen our understanding of the origins and rules of life. However, the rapid decline in biodiversity reported recently foreshadows a potentially catastrophic collapse of many important ecosystems and the associated irreversible loss of many forms of life on our planet. Immediate action by conservationists of all stripes is required to avert this disaster. In this Spotlight, we draw together insights and proposals discussed at a recent workshop hosted by Revive & Restore, which gathered experts to discuss how stem cell technologies can support traditional conservation techniques and help protect animal biodiversity. We discuss reprogramming, in vitro gametogenesis, disease modelling and embryo modelling, and we highlight the prospects for leveraging stem cell technologies beyond mammalian species

Refrigerated Storage of Red Deer Epididymal Spermatozoa in the Epididymis, Diluted and with Vitamin C Supplementation

P. 212-220We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti‐oxidant. Twenty‐nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 × 106 sperm/ml in Tris‐citrate‐fructose with 20% egg yolk. Control group was stored as such, and Anti‐oxidant group was supplemented with 0.8 mm vitamin C. The remaining epididymides and the diluted samples were stored at 5°C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer‐assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.S

Advancing stem cell technologies for conservation of wildlife biodiversity.

Wildlife biodiversity is essential for healthy, resilient and sustainable ecosystems. For biologists, this diversity also represents a treasure trove of genetic, molecular and developmental mechanisms that deepen our understanding of the origins and rules of life. However, the rapid decline in biodiversity reported recently foreshadows a potentially catastrophic collapse of many important ecosystems and the associated irreversible loss of many forms of life on our planet. Immediate action by conservationists of all stripes is required to avert this disaster. In this Spotlight, we draw together insights and proposals discussed at a recent workshop hosted by Revive & Restore, which gathered experts to discuss how stem cell technologies can support traditional conservation techniques and help protect animal biodiversity. We discuss reprogramming, in vitro gametogenesis, disease modelling and embryo modelling, and we highlight the prospects for leveraging stem cell technologies beyond mammalian species

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09 S and 48°26'42 W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were washed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mineral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham's F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of morulae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.Do Trópico de Capricórnio ao Equador, sabe-se que a sazonalidade no gato domestico é ausente, i.e., estes animais são considerados reprodutores não sazonais nestas regiões. Nós hipotetizamos que esta particularidade possa ter alguma influência sobre a produção embrionária in vitro. O objetivo deste experimento foi determinar a porcentagem de clivagem e formação de mórulas e blastocistos produzidos a partir de oócitos recuperados de ovários de gatas em três condições - folicular, lútea ou inativa - durante duas estações reprodutivas pelas quais gatas passam na região sudeste do Brasil (22°53'09 S e 48°26'42 O) - estação não reprodutiva (ENR), que compreende os meses de janeiro a março; e estação reprodutiva (ER), agosto à outubro. Trinta gatas foram castradas. Os ovários foram classificados de acordo com sua condição e foram fatiados em PBS para liberação dos complexos cumulus oophorus (COC). COC grau I foram lavados três vezes em H-MEM suplementado com BSA, glutamina, piruvato sódico, cisteína, estreptomicina e penicilina. Os oócitos foram incubados em grupos de 20-30 em 400 µL de DMEM suplementado com FSH, LH, estradiol, IGF-I e fator de crescimento fibroblástico básico sob óleo mineral por 30 ou 36 horas em atmosfera úmida de 5% de O2, 5% CO2 e 90% N2 a 38°C. Os COC foram fertilizados em meio Ham's F-10 suplementado com BSA, cisteína, piruvato e estreptomicina/penicilina (meio de cultura) com sêmen fresco selecionado através da técnica de swim up. Dezoito horas depois, os presumíveis zigotos foram denudados, a porcentagem de clivagem foi determinada e cada 10 zigotos foram transferidos para gotas de 100 mL de meio de cultura para cultivo durante 3 dias. Após 72 horas de cultivo, a porcentagem de formação de mórulas foi avaliada e estas estruturas foram transferidas para gotas do mesmo meio de cultivo. No oitavo dia de cultivo a formação de blastocisto foi avaliada. Durante a ENR, de um total de 272 (inativo), 162 (lútea) e 134 (folicular) oócitos fertilizados, a porcentagem de clivagem de zigotos, formação de mórulas e de blastocistos derivados de ovários inativos foi 24,63, 16,54 e 8,09 respectivamente; para aqueles oriundos de ovários na condição lútea, a porcentagem foi de 21,6, 12,96 e 8,64, e para aqueles provenientes de ovários na fase folicular, foi de 24,62, 16,41 e 8,21. Considerando a ER, de um total de 102 (inativo), 198 (lútea) e 86 (folicular) oócitos fertilizados, a frequência relativa (%) de zigotos clivados, mórulas e blastocistos derivados de ovários na condição inativa foi de 64,7, 41,17 e 23,53 respectivamente; para aqueles oriundos de ovários na condição lútea, a porcentagem foi de 64,14, 40,41 e 23,73, e para aqueles provenientes de ovários na fase folicular, foi de 63,95, 39,54 e 24,41. Os resultados deste experimento demonstraram que não houve diferença estatística significante (P < 0.05) na frequência de embriões clivados e na formação de mórulas e blastocistos quando comparadas as três condições ovarianas dentro da mesma estação. Entretanto, a ER apresentou resultados melhores considerando as taxas de clivagem e formação de mórula e de blastocisto se comparada à ENR.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de Mato Grosso Laboratório de Reprodução AnimalUniversidade de São Paulo Faculdade de Medicina Veterinária e ZootecniaUniversidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Departamento de Reprodução Animal e Radiologia VeterináriaUniversidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Departamento de Reprodução Animal e Radiologia VeterináriaFAPESP: 05/54832-