Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii)

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski’s horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski’s horse spermatozoa during liquid storage at 4 °C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski’s versus domestic horse. When Przewalski’s horse spermatozoa were incubated at 4 °C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P < 0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P > 0.05) between the Przewalski’s (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose–EDTA–glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondrial membrane potential were 42.4%, 21.8%, 88.7% and 25.4 CN (CN = mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalski’s and 49.3%, 24.6%, 88.9% and 25.8 CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P > 0.05) among treatments across species, mitochondrial membrane potential was higher (P < 0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalski’s stallion sperm expressed higher (P < 0.05) post-thaw total motility in BOTU and SM compared to EQ, whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalski’s stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalski’s and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalski’s horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF.     

Lessons from biodiversity—the value of nontraditional species to advance reproductive science,conservation, and human health

Reproduction is quintessential to species survival. But what is underappreciated for this discipline is the wondrous array of reproductive mechanisms among species— variations as diverse as the morphology of the species themselves (more than 55,000 vertebrate and 1.1 million invertebrate types). We have investigated only a tiny fraction of these species in reproductive science. Besides the need to fill enormous gaps in a scholarly database, this knowledge has value for recovering and genetically managing rare species as well as addressing certain reproductive issues in humans. This article provides examples, first to advise against oversimplifying reproduction and then to show how such knowledge can have practical use for managing whole animals, populations, or even saving an entire species. We also address the expected challenges and opportunities that could lead to creative shifts in philosophy and effective actions to benefit more species as well as a future generation of reproductive scientists. Mol. Reprod. Dev. 77: 397–409, 2010. © 2009 Wiley‐Liss, Inc.     

Retention of plastic-tipped dart tags in African tigerfish Hydrocynus vittatus

Estimates of tag retention and tagging-related mortality are essential for mark-recapture experiments. Mortality and tag loss were estimated from 15 tigerfish Hydrocynus vittatus marked using Hallmark model PDL plastic-tipped dart tags released into a 1 730 m² pond at Kamutjonga Inland Fisheries Institute, Namibia, and inspected bi-monthly for the presence or absence of tags. No mortality was observed during the experiment. All marked fish had lost their tags after 10 months and 50% tag loss was estimated at 3.9 months. The high tag loss rate indicates that PDL plastic-tipped dart tags are not suitable for long-term studies on this species.     

Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocytes

Ability of ovarian oocytes from the domestic dog to complete nuclear maturation in vitro (IVM) varies markedly among donors and generally is 20% or less of all oocytes cultured. To identify the cause(s) underlying these significant variations in meiotic maturation (to metaphase II; MII), we retrospectively analyzed data from 1,643 oocytes recovered from 90 bitches for which stage of reproduction and season of year were known. Neither stage of reproduction (proestrus/estrus, diestrus, anestrus, or prepuberty) nor season (P > 0.05) influenced the ability of oocytes to achieve nuclear maturation in vitro. A second study was conducted to examine the impact of follicular size on meiotic maturation. Populations of large oocytes were recovered from four categories of follicles (ranging from <0.5 to > 2 mm in diameter) and cultured in TCM 199 for 48 hr. Follicular size influenced (P < 0.05) meiotic competence. Mean percentages of MII oocytes were 16.9 +/- 9.2, 26.1 +/- 7.6, 38.4 +/- 9.2, and 79.5 +/- 10.9 for oocytes recovered from < 0.5 mm, > or = 0.5-< 1 mm, 1-2 mm, and > 2 mm diameter follicles, respectively. In summary, stage of reproduction and season have no impact on the ability of dog oocytes to achieve nuclear maturation in vitro. However, we demonstrated for the first time that dog oocytes acquire meiotic competency during follicular development. IVM success of selected oocytes from large size follicles (almost 80%) is about 60% higher than measured in most previous studies involving randomly collected oocytes.     

Requirement for, and patterns of, pyruvate and glutamine metabolism in the domestic dog oocyte in vitro

Supplementation of energy substrates to culture medium is essential for resumption and completion of meiosis in vitro for many mammalian species. Objectives were to study the dog oocyte, specifically the influences of pyruvate and glutamine on maturation and the utilization of these two substrates at various developmental stages and incubation times. Ovarian oocytes (n=681) were obtained from spayed bitches and cultured for 48 hr in TCM 199 medium containing various concentrations of pyruvate (0-2.5 mM) and glutamine (0-4 mM) before being assessed for nuclear status. For analyzing metabolic activity, 259 dog oocytes were cultured for 0, 12, 24, 36, or 48 hr, assessed for pyruvate and glutamine metabolism using the hanging drop method and then evaluated for nuclear status. Neither pyruvate nor glutamine had influence (P > 0.05) on oocyte maturation in vitro (IVM). However, both culture interval and meiotic status influenced pyruvate uptake (P < 0.05). Specifically, pyruvate uptake declined as the oocyte progressed from the germinal vesicle (GV) to metaphase II (MII) stage. Glutamine oxidation decreased as culture duration progressed (P < 0.05). In summary, pyruvate or glutamine is not required to promote successful IVM of dog oocytes. But, both substrates are being metabolized, and in patterns different to the domestic cat, another carnivore species. Pyruvate played an important role earlier in the maturational process, and less glutamine was oxidized as the oocyte neared nuclear maturation. These variations emphasize the importance of defining species specificities in carnivores before expecting consistently successful IVM/IVF.     

Factors affecting captive whooping crane egg fertility: A retrospective analysis

The whooping crane (Grus americana) has been managed in captivity since the 1960s following a substantial genetic bottleneck in the wild population. Through major ex situ conservation efforts, there are 158 whooping cranes managed in North American institutions and chicks are released annually into the wild. Current reintroduction goals for the whooping crane however, are impeded by poor reproduction within the ex situ population, in part because of low egg fertility. Development of improved management techniques to overcome low egg fertility requires a better understanding of factors that influence egg fertility. We collected data for eggs laid at Patuxent Wildlife Research Center from 2005–2014 (n = 438 eggs; n = 23 pairs). We constructed 5 sets of generalized linear mixed-models, with a Bernoulli-distributed response variable (fertile or infertile), to address the effects of egg-specific variables (month laid, sequence, and clutch order of the egg), life-history events of the male and female (age, rearing method, wing condition, age at first pairing, age at current pairing, and female age at first laying), pair-specific characteristics (kinship, years paired, chick-rearing experience, and previous pairings), and captive management decisions (inclusion in the artificial insemination program and pair experience chick-rearing) on the probability of egg fertility. Our results indicate that female-specific factors (especially age, age at current pairing, and wing status) and pair-specific factors of kinship, chick-rearing experience, copulation, and inclusion in the artificial insemination program influenced fertile egg production. Specifically, the younger a female is when paired with her current social mate, the higher probability that her eggs will be fertile. Furthermore, high kinship reduced fertility, whereas chick-rearing experience and artificial insemination of females with a donor male other than her social mate increased fertility. Further research on mate selection and reproductive mechanisms is needed to better understand egg laying and egg fertility in the whooping crane. © 2019 The Wildlife Society.     

Natural history of SLC11 genes in vertebrates: tales from the fish world

Background  

The SLC11A1/Nramp1 and SLC11A2/Nramp2 genes belong to the SLC11/Nramp family of transmembrane divalent metal transporters, with SLC11A1 being associated with resistance to pathogens and SLC11A2 involved in intestinal iron uptake and transferrin-bound iron transport. Both members of the SLC11 gene family have been clearly identified in tetrapods; however SLC11A1 has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. In the present work we characterized the SLC11 genes in teleosts and evaluated if the roles attributed to mammalian SLC11 genes are assured by other fish specific SLC11 gene members.     

Activity pattern, reproductive behaviors and gonadal hormones in the raccoon dog (Nyctereutes procyonoides)

The objectives of this study were to (1) assess year-round behaviors and activity patterns of captive raccoon dogs (Nyctereutes procyonoides) and (2) characterize the species' reproductive endocrinology. Behaviors and activity patterns of 12 (5.7) animals were recorded over a 1-year period. During that time, fecal samples were collected 2-7 times/week from 16 (7.9) individuals (six of these were included in the behavioral study) for the analysis of testosterone, progesterone and estrogen metabolite concentrations. Activity pattern and excretion of gonadal steroids followed a seasonal pattern. Specifically, dogs were cathemeral in summer, and primarily nocturnal in winter. In the males, testosterone concentrations were at baseline from April through September, began to rise in October and reached peak concentrations in February (P<0.05). In the females, elevated estrogen (P<0.05) was observed in March followed by an increase in progestagen concentrations from March through May (P<0.05) in both pregnant and pseudopregnant animals. Gender significantly influenced monthly testosterone/estrogen ratio (P<0.01); values were higher in males than in females throughout the year with overall percentage of overlapping values between males and females being 28%. In summary, this study characterized cirannual fluctuations in behaviors and gonadal steroid metabolites in the raccoon dog maintained in captivity. Because there is no obvious sexual dimorphism, the differences in testosterone/estrogen ratio may be useful for gender differentiation (72% accuracy), especially among individuals living in the wild.     

Occurrence of Three Felids across a Network of Protected Areas in Thailand: Prey,Intraguild, and Habitat Associations

Clouded Leopard, Leopard, and Tiger are threatened felids in Southeast Asia, but little is known about the factors influencing their distributions. Using logistic regression, we assessed how habitat variables, prey detection patterns, and presence of intraguild predators affect the occurrence of these felids across 13 protected areas within Thailand. Our analysis is based on data from 1108 camera‐trap locations (47,613 trap‐nights). Clouded Leopard and Leopard are associated with habitat where Red Muntjac and Eurasian Wild Pig were most likely to be present. Tiger are associated with habitat with a higher likelihood for the presence of Gaur, Eurasian Wild Pig, and Sambar. Clouded Leopard and Tiger were both weakly associated with areas with mature evergreen forest. Besides availability of prey, associations with potential competitors also appear to influence the distribution of these felids, although the strength of these effects requires further investigation. Occurrence rates for Clouded Leopard were no different in protected areas with Leopard versus without Leopards. Leopard had similar occurrence rates regardless of the presence of Tiger, but Leopards were less likely to be detected at the same camera‐trap points with the larger felid. Our results suggest that the two most commonly photographed prey species in the study areas serve as key prey species, Eurasian Wild Pig for all three carnivores and Red Muntjac for Leopard and Clouded Leopard.