Characteristics of high-quality Asian elephant (Elephas maximus) ejaculates and in vitro sperm quality after prolonged chilled storage and directional freezing.

The in vitro quality of spermatozoa from one elephant (Elephas maximus) was examined after chilled storage and directional freezing (DF). High-quality, non-contaminated ejaculates (77.6±6.0% progressive motility, 3.9±1.5 µg creatinine mL(-1) raw semen, 2.7±0.6% detached heads) were cryopreserved after 0 (0hStor), 12 (12hStor) and 24 h (24hStor) of chilled storage. At 0 h and 6h post-thawing, total motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and normal morphology were similar (P>0.05) across treatments. In contrast, progressive motility, rapid velocity and several kinematic parameters were lower (P<0.05) for 24Stor compared with 0hStor at 0 h post-thaw. By 6 h post-thaw, amplitude of lateral head displacement and velocity parameters (average pathway, straight-line and curvilinear velocity) were lower (P<0.05) for 24hStor compared with 0hStor and 12hStor. DNA integrity was high and remained unchanged (P>0.05) across all groups and processing stages (1.6±0.6% of cells contained fragmented DNA). Results indicate that DF after up to 12 h of chilled storage results in a post-thaw sperm population of acceptable quality for artificial insemination. These findings have implications for the cryopreservation of sex-sorted spermatozoa, which typically undergo more than 12 h of chilled storage prior to sorting and preservation.

Evaluation of motility, membrane status and DNA integrity of frozen-thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation.

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Thyroid hormone concentrations associated with age, sex, reproductive status and apparent reproductive failure in the Amazon river dolphin (Inia geoffrensis).

This study was conducted to characterize immunoreactive thyroid hormone concentrations in wild Amazon river dolphins, also called boto (Inia geoffrensis) by age group, sex, pregnancy and lactation status, and to determine if thyroid hormone concentration differences could be detected between pregnant females with and without successful parturition outcomes. Radioimmunoassays were used to analyse total T 3 and total T 4 in 182 serum samples collected from 172 botos living in the Mamirauá Sustainable Development Reserve, in the Brazilian Amazon from 2003 through 2015. Age significantly affected tT 3 and tT 4 concentrations in males, with values in immature males and females being significantly lower than those in adult males, whereas no age effects were noted between immature females and adult non-pregnant, non-lactating females. Significant sex differences were noted in tT 3 concentrations between immature males and females and in tT 4 concentrations between adult males and females. These resulted in significant differences in the tT 3:tT 4 ratio between males and females within the immature and adult groups. Lactating and non-pregnant adult females had significantly higher tT 3 concentrations than pregnant females, and this difference was primarily driven by a 12% drop in tT 3 concentrations during the last two-thirds of pregnancy. No differences in thyroid hormone concentrations were detected between females diagnosed as pregnant and later found to have or not have a live calf. These results are the first to define thyroid hormone reference intervals and normal physiological variations in a wild population of river dolphins.

Amphiphilic block copolymers as flexible membrane materials generating structural and functional mimics of green bacterial antenna complexes.

We describe the ability of a short-chain amphiphilic block copolymer to self-assemble to form an artificial supramolecular light-harvesting system. Specifically, we demonstrate that the 2.5 kDa, poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD), exhibits sufficient morphological flexibility as a membrane material and enables generation of mimics of three-dimensional chlorosomes as well as supported membrane bilayers containing energy acceptors. This overall architecture replicates green bacterial light-harvesting function whereby these assemblies exhibit long-range order and three-dimensional morphology similar to native chlorosomes and are capable of energy transfer internally and to external acceptors, located in a supporting biomimetic polymer membrane. Unlike native green bacterial systems that use multiple lipids as a matrix to generate the appropriate environment for chlorosome assembly and function, the described system matrix is comprised entirely of a single polymer amphiphile. This work demonstrates the potential of short-chain amphiphilic block copolymers in generating self-assembled, bio-mimetic membrane architectures, and in doing so, generates scalable, spatial-energetic landscapes for photonic applications. Finally, the results presented provide evidence of minimal requirements to induce chlorosome-like organization and function.

Sperm sex-sorting and preservation for managing the sex ratio and genetic diversity of the southern white rhinoceros (Ceratotherium simum simum).

White rhinoceros ejaculates (n=9) collected by electroejaculation from four males were shipped (10°C, 12h) to develop procedures for the production of chilled and frozen-thawed sex-sorted spermatozoa of adequate quality for artificial insemination (AI). Of all electroejaculate fractions, 39.7% (31/78) exhibited high quality post-collection (≥70% total motility and membrane integrity) and of those, 54.8% (17/31) presented reduced in vitro quality after transport and were retrospectively determined to exhibit urine-contamination (≥21.0µg creatinine/ml). Of fractions analyzed for creatinine concentration, 69% (44/64) were classified as urine-contaminated. For high quality non-contaminated fractions, in vitro parameters (motility, velocity, membrane, acrosome and DNA integrity) of chilled non-sorted and sorted spermatozoa were well-maintained at 5°C up to 54h post-collection, whereby >70% of post-transport (non-sorted) or post-sort (sorted) values were retained. By 54h post-collection, some motility parameters were higher (P<0.05) for non-sorted spermatozoa (total motility, rapid velocity, average path velocity) whereas all remaining motion parameters as well as membrane, acrosome and DNA integrity were similar between sperm types. In comparison with a straw method, directional freezing resulted in enhanced (P<0.05) motility and velocity of non-sorted and sorted spermatozoa, with comparable overall post-thaw quality between sperm types. High purity enrichment of X-bearing (89±6%) or Y-bearing (86±3%) spermatozoa was achieved using moderate sorting rates (2540±498X-spermatozoa/s; 1800±557Y-spermatozoa/s). Collective in vitro characteristics of sorted-chilled or sorted-frozen-thawed spermatozoa derived from high quality electroejaculates indicate acceptable fertility potential for use in AI.

Development and evaluation of deep intra-uterine artificial insemination using cryopreserved sexed spermatozoa in bottlenose dolphins (Tursiops truncatus).

Since its development in bottlenose dolphins, widespread application of AI with sex-selected, frozen-thawed (FT) spermatozoa has been limited by the significant expense of the sorting process. Reducing the total number of progressively motile sperm (PMS) required for an AI would reduce the sorting cost. As such, this research compared the efficacy of small-dose deep uterine AI with sexed FT spermatozoa (SEXED-SMALL; ~50×10(6)PMS, n=20), to a moderate dose deposited mid-horn (SEXED-STD, ~200×10(6)PMS; n=20), and a large dose of FT non-sexed spermatozoa deposited in the uterine body (NONSEXED-LARGE, 660×10(6)PMS, n=9). Ten of the 11 calves resulting from use of sexed spermatozoa were of the predetermined sex. Similar rates of conception (NONSEXED-LARGE: 78%, SEXED-STD: 60%, SEXED-SMALL: 57%) and total pregnancy loss (TPL: NONSEXED-LARGE: 28.6%; SEXED-STD: 41.0%; SEXED-SMALL: 63.6%) were observed across groups, but early pregnancy loss (EPL,

Sperm DNA fragmentation and morphological degeneration in chilled elephant (Elephas maximus and Loxodonta Africana) semen collected by transrectal massage.

Ejaculates from nine Asian and two African elephants were analysed to gain a further understanding of mechanisms underlying variable semen quality after transrectal massage. Semen analysis was performed after collection (0 h; subjective motility parameters only) and after 24 h of chilled storage at 10 °C (24 h; all ejaculate and sperm characteristics). Ejaculates with ≤50% total motility (TM) at 24 h, which represented >90% of collection attempts, contained a sperm population with a high degree of DNA damage (64.2 ± 19.2% fragmented DNA) and an elevated incidence of detached heads (43.3 ± 22.5%). In contrast, good quality ejaculates designated as those with >50% TM at 24 h displayed higher (p < 0.05) values of sperm kinetic parameters, DNA integrity and normal morphology. Fertility potential was high for good quality ejaculates from two males (one Asian and one African bull) based on in vitro characteristics after chilled storage for up to 48 h post-collection. Urine contamination of semen, as assessed quantitatively by creatinine concentration, was confirmed as a significant factor in reduced elephant ejaculate quality. However, the identification of considerable DNA damage and morphological degeneration in the majority of ejaculates after only 24 h of chilled storage indicates that sperm ageing could be a primary contributor to inconsistent semen quality in the elephant.

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas).

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean +/- SD) 73.0 +/- 12.2, 38.4 +/- 8.8, 88.0 +/- 0.1, and 59.3 +/- 15.7%, respectively. A series of GnRH injections (3 x 250 microg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 +/- 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 +/- 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 +/- 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 +/- 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 +/- 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.